Genetic test for α-mannosidosis

ABSTRACT

A method for diagnosing or screening for bovine α-mannosidosis comprises detecting in nucleic acid samples from cattle the presence or absence of α-mannosidosis-causing mutations in the gene encoding bovine lysosomal α-mannosidase (LAMAN). A method of detecting α-mannosidosis-causing mutations in cattle comprises detecting the presence or absence of base transitions in the gene encoding bovine LAMAN which are associated with the disease.

The present invention relates to α-mannosidosis and its detection, inparticular the detection of bovine α-mannosidosis.

α-mannosidosis is an autosomal, recessively inherited lysosomal storagedisorder that has been clinically well characterised (M. A. Chester etal., 1982, in Genetic Errors of Glycoprotein Metabolism pp 90-119,Springer Verlag, Berlin). It is a very common disease in cattle but hasalso been found in man and cat. Glycoproteins are normally degradedstepwise in the lysosome and one of the steps, namely the cleavage ofα-linked mannose residues from the non-reducing end during the ordereddegradation of N-linked glycoproteins is catalysed by the enzymelysosomal α-mannosidase (EC 3.2.1.24). However, in α-mannosidosis, adeficiency of the enzyme α-mannosidase results in the accumulation ofmannose rich oligosaccharides. As a result, the lysosomes increase insize and swell, which impairs cell functions.

In man, the disease is rare. The symptoms of α-mannosidosis includepsychomotor retardation, ataxia, impaired hearing, vacuolizedlymphocytes in the peripheral blood and skeletal changes. (Chester et alsupra).

In cattle the disorder is much more common and results in mentalretardation, skeletal changes, ataxia, a fine head tremor, aggressivebehaviour and premature death. The disease has been reported amongcattle in both Northern and Southern Hemispheres and in different breedsof cattle. Among cattle of the Angus breed there is a variation inphenotypic expression spanning from death within days to slowlyprogressing neurological deterioration that lasts for months (Healy etal., 1990, Res. Vet. Sci., 49: 82-84). Among other breeds of cattle suchas Galloway, the symptoms can be more severe resulting in still birth ordeath within a few days after birth (Healy et al supra). As mentionedabove, α-mannosidosis is widespread in cattle and consequently is ofconsiderable economic importance.

Currently, no treatment of the disease is available and consequently,not only are diseased cattle lost with respect to meat production, butit is important to prevent spread of the disease in breeding programmes.There is therefore a need to detect cattle that have or are carriers ofα-mannosidosis. A known method of detection has been to determine theenzyme activity of lysosomal α-mannosidase. It is generally decreased to40% of normal activity among cattle carrying α-mannosidosis (ie.heterozygotes) and less than 10% in cattle exhibiting fullα-mannosidosis (ie. homozygotes). However, activity levels may varybetween individuals and it sometimes becomes difficult to distinguishnormal cattle from heterozygous carriers. This severely limits theutility of the enzyme test in detecting carriers.

The enzyme activity assay requires withdrawal of a blood sample lessthan 24 hours before analysis is begun. The enzyme activity isdetermined in isolated granulocytes (Healy, 1981, Res. Vet. Sci., 30:281-283) so it is first necessary to isolate and purify the white bloodcells. The enzyme activity is determined using the calorimetricsubstance p-nitrophenyl alpha D-mannopyranoside at pH 3.7 or 4-methylumbelliferyl alpha D-mannopyranoside. This enzymatic test is verysensitive, contamination of the cells can result in erroneous resultsand in some instances it might be difficult to distinguish betweenheterozygous and normal values of α-mannosidase activity.

The fact that only a short time can lapse between the taking of thesample from the animal and laboratory analysis means significant expenseis incurred in ensuring speedy delivery to the laboratory and once atthe laboratory samples have to be processed immediately, with the use oftime consuming cell isolation techniques. The blood sample must usuallybe taken by a vet adding to the cost of the test. Thus, it will be seenthat the disadvantages of the enzyme test are not inconsiderable andthere remains a need for a straightforward and inexpensive text todetect cattle that have or are carriers of α-mannosidosis.

The nucleotide sequence of human lysosomal α-mannosidase has beendetermined (Nebes and Schmidt, 1994, Biochem. Biophys. Res. Comm., 200:239-245) and a mutation causing α-mannosidosis in humans identified,namely a base transition results in a His to Leu replacement in aconserved region of the gene for α-mannosidase (Tollersrud et al., 1995,10th ESGLD Workshop, Cambridge, England). However, in the case ofcattle, full sequence information for the gene encoding bovine lysosomalα-mannosidase (LAMAN) gene is not available, and more significantly, acorresponding mutation in the gene encoding LAMAN has not up to now beenknown. Consequently no gene based test for the disease in cattle has upto now been available.

Mutations in the gene encoding bovine LAMAN which cause α-mannosidosisin cattle have now been elucidated, enabling and leading to thedevelopment of a genetic test for the disease.

Thus, in one aspect, the present invention provides a method fordiagnosing or screening for bovine α-mannosidosis, comprising detecting,in nucleic acid samples from cattle, the presence or absence ofα-mannosidosis-causing mutations in the gene encoding bovine LAMAN.

The present invention thus provides a method which not only enables theready diagnosis of diseased cattle, but also permits cattle to bescreened for the presence of the disease-causing allele of theLAMAN-encoding gene, enabling carriers to be detected and removed frombreeding programmes. As used herein, the term “screening” thus includesdetecting the presence of mutated LAMAN-encoding alleles in healthycattle, ie. carriers, as well as in diseased animals. In this aspect,the invention can thus be seen to provide a genetic test for detectingthe disease-causing α-mannosidase gene in cattle.

As mentioned above, our studies have shown that point mutations in theLAMAN gene can be identified which are associated with the α-mannosidasephenotype. Such α-mannosidosis-causing mutations are thus basetransitions in the LAMAN-encoding gene, leading to amino acidsubstitutions in the encoded LAMAN protein.

In a further aspect, the present invention thus provides a method ofdetecting α-mannosidosis-causing mutations in cattle, comprisingdetecting the presence or absence of base transitions in the geneencoding bovine LAMAN, which are associated with disease.

As used herein the term “associated with disease” means that the basetransitions result, as discussed above, in amino acid substitutions inthe LAMAN protein which affect the functioning of the enzyme such thatin homozygotes, α-mannosidosis is caused.

As will be described in more detail in the Examples below, in workleading to the present invention, bovine LAMAN has been purified andbeen found to be encoded by a single gene. A cDNA encoding the bovineLAMAN protein has been prepared and sequenced (SEQ ID No. 1) and isshown in FIG. 1, together with its amino acid translation in singleletter code. Genomic sequencing studies of the bovine LAMAN-encodinggene have also been undertaken and a partial genomic sequence is shownin FIG. 2 (SEQ ID No. 3).

Such sequences represent further aspects of the invention. Thus, in afurther aspect, the present invention can be seen to provide a nucleicacid molecule comprising all or a portion of a nucleotide sequence asshown in any one of FIG. 1 or 2 (SEQ. ID Nos. 1 or 3) or a sequencewhich is complementary thereto, or which is a degenerate or allelicvariant thereof, or a substantially homologous sequence having at least85%, preferably at least 90% sequence identity therewith.

A still further aspect of the invention provides the use of a nucleicacid molecular comprising a nucleotide sequence as shown in FIG. 1 or 2or (SEQ ID Nos. 1 or 3) a sequence which is complementary thereto, orwhich is a degenerate or allelic variant thereof, or is a substantiallyhomologous sequence having at least 85%, preferably at least 90%sequence identify therewith, or a part of any said sequence, in thedetection of α-mannosidosis-causing mutation in the gene encoding bovineLAMAN.

RNA transcripts of the above-mentioned nucleotide sequences may also beused.

Sequencing of the bovine LAMAN cDNA was carried out by the use of PCRtechniques on DNA extracted from a bovine cDNA library. Details,including the primers used, are given in the Examples below. cDNA fromnormal and affected cattle were compared by direct sequencing of PCRproducts and a number of different breeds were studied. This enabled usto determine that α-mannosidosis in different cattle breeds may beassociated with different mutations, ie. different base transitions, atdifferent positions in the bovine LAMAN gene, leading to different aminoacid replacements or substitutions in the LAMAN protein. The presentinvention thus encompasses the detection of a range of differentmutations ie. point mutations or base transitions in the bovine LAMANgene, which may be associated with α-mannosidosis, and which may vary,both within breeds, and from breed to breed. Thus, for example, thebreeds Angus, Murray Grey (which is a breed derived from Angus) andGalloway were studied, these representing breeds of economic importanceand wide distribution.

In cDNA from the Angus breed of cattle, a single nucleotide substitutioncytosine (C) for thymine (T), was identified that was predicted toresult in the substitution of the amino acid leucine for phenylalanine.The particular phenylalanine residue is conserved between a number ofdifferent species indicating an important physiological function. It wasfound that the T to C substitution was conserved in both alleles of 3Angus cattle affected by α-mannosidosis, in one of the alleles of 12carriers of the disease and in no alleles of 58 normal Angus cattle.Similar results have been observed with Murray Grey and Red Anguscattle. Thus, we have found that the base transition ie. of the cDNAencoding bovine LAMAN, T to C at position 961 (T961C) of FIG. 1(position 975 of SEQ ID No. 1), leading to an amino acid substitution ofPhenylalanine (Phe) to Leucine (Leu) at amino acid position 321 isassociated with the α-mannosidase-causing genotype in Angus andAngus-derived breeds of cattle, such as Murray Grey, Red Angus andBrangus.

In another breed of cattle, Galloway, comparison of the sequences ofnormal LAMAN cDNA and cDNA from a carrier of α-mannosidosis alsorevealed a base transition at a single position, but different from theposition in Angus. In Galloway cattle there appears to be a G to Atransition at position 662 of FIG. 1 (position 677 of SEQ ID No. 1),leading to an amino acid substitution of Arginine (Arg) to Histidine(His) at position 221. Only Arginine or Lysine appear at this positionin the particular class of α-mannosidase, indicating that these closelyrelated side chains are physiologically important in this position. TheG to A transition was found in both alleles of 2 affected Gallowaycattle, in one of the alleles of each of 7 carriers and in no alleles of29 normal Galloway cattle. Thus, in the Galloway breed of cattle, the Gto A transition at position 662 (G662A) of FIG. 1 (SEQ ID No. 1) appearsto be primarily associated with α-mannosidosis.

Thus, in a particular aspect, according to the invention there isprovided a DNA molecule which codes for bovine LAMAN as hereinbeforedefined, wherein the nucleotide at position 961 of FIG. 1 (SEQ ID No. 1)is cytosine. Also provided, is a DNA molecule coding for bovine LAMAN,wherein the nucleotide at position 662 of FIG. 1 (SEQ ID No. 1) isadenine.

In a preferred embodiment, the present invention also provides methodsof diagnosis or screening, and methods of detection as hereinbeforedefined, wherein the base transitions T to C at position 961 of FIG. 1(SEQ ID No. 1) (T961C) and/or G to A at position 662 of FIG. 1 (SEQ IDNo. 1)(G662A) are detected.

Having identified mutations causing α-mannosidosis in cattle,straightforward gene based tests may be developed for identifying themutant alleles, particularly in carriers which have one normal unmutatedallele for the LAMAN gene. At their broadest, as mentioned above, suchtests involve the detection of the aforementioned alleles. Such tests,which are exemplified in more detail below, have been developed andconstitute further aspects of the present invention.

The present gene based detection methods which form aspects of thepresent invention have advantages over the earlier enzyme assays, asonly very small amounts of starting material are required. For example,hair roots contain sufficient DNA for mutation detection studies. Othersources of DNA for the detection method include semen. Moreover, as DNAis relatively stable the samples may be sent via ordinary mail to thelaboratories for testing and can be stored for several months below −20°C. without degradation of the DNA. The DNA based detection method ismore reliable than the enzymatic test for detection of heterozygosity;the enzyme activity in carriers and normal cattle may overlap, whereasthe frequency of mutated alleles in a heterozygous subject is strictly50%. Those cattle homozygous for the mutated allele are likely toexhibit symptoms which can be observed by eye and it is therefore thedetection of heterozygous carriers which is most important. Nucleic acidbased detection methods also minimise the risk of contamination, forexample from viruses which may be found in blood samples.

The methods of the invention may be carried out on any nucleic acidcarrying or containing sample from cattle, including any such tissue orbody fluid sample. Thus while samples such as blood, or blood derivedsamples may be used, the tests may conveniently be carried out asreadily and easily obtainable samples such as semen, saliva or hairroots. The nucleic acid for analysis may be extracted from such samplesusing standard extraction or isolation techniques which are well knownand widely described in the literature (see for example Sambrook et al.,1989, in Molecular Cloning: A Laboratory Manual, 2nd edition). Thenucleic acid may be RNA or DNA, but conveniently will be DNA, and may besubjected to amplification using eg. any of the widely available invitro amplification techniques.

Any method of detecting the disease-causing mutations ie. basetransitions may be used according to the invention, and many suitablemethods are described in the literature. Thus, for example, any methoddescribed in the literature for detecting known point mutations may beused.

A test which includes the amplification of a DNA fragment by PCR hasbeen developed. Oligonucleotide primers that are complementary to DNAsequences upstream and downstream of the α-mannosidosis causing mutationare synthesised. These primers are used to amplify the DNA region thatis located between them. Due to the amplification step, small amounts ofstarting material may be employed, for example, hair roots containsufficient DNA for mutation detection studies.

As mentioned above, the mutation can be detected by a number oftechniques known in the art which include DNA sequencing; cleavage ofRNA-DNA or RNA-RNA hybrids at mutation sites using RNase; mis-matchdetection using heteroduplex analysis; detection of mutations bysingle-strand conformation polymorphism analysis; detection of mutationsby denaturing gradient gel electrophoresis; chemical or enzymaticcleavage of heteroduplex DNA to identify mutations and detection ofmutations by restriction fragment length polymorphism (RFLP).

In the case of the α-mannosidosis-causing mutations which have beenidentified according to the present invention, a single base issubstituted in an allele which codes for the substantially inactive (ie.mutated) form of α-mannosidase. This base transition causes a change inthe number of restriction sites in the gene, so the digest resultingfrom treatment with a particular restriction enzyme will vary betweennormal and mutated alleles. Thus for example, in the case of the Angusand Angus-related breeds the T 961 C substitution results in thecreation of an extra site for the restriction enzyme Mnl I, which may beused to distinguish the normal and mutant alleles, since the mutantsequence will be cleaved at this site by Mnl I, whereas the normalsequence will not, resulting in a different restriction digestionpattern. FIG. 3 shows the different fragments that result when part ofthe α-mannosidase gene in Angus cattle is treated with the restrictionenzyme Mnl I.

In the case of the Galloway breed, the G 662 A substitution results inthe loss of a Bsa HI restriction site. Thus Bsa HI digestion will give adifferent restriction pattern as between normal and mutant alleles,since in this case the normal sequence will be cleaved at this site byBsa HI, whereas the mutant sequence will not. Therefore, a furtheraspect of the present invention provides a method of diagnosing orscreening for bovine α-mannosidosis comprising detecting the presence orabsence of α-mannosidosis-causing mutations in the gene encoding bovineLAMAN using a restriction enzyme.

In view of the fact that restriction sites may conveniently be createdor removed in the α-mannosidosis causing mutant alleles of the geneencoding bovine LAMAN, detection by RFLP represents a particularlypreferred method and comprises a further aspect of the presentinvention. PCR-based RFLP analysis was developed to provide anon-radioactive method for fast and simple detection of mutations. Thismethod relies upon the introduction of restriction endonuclease sitespermitting differential cleavage between normal and mutant sequences.The α-mannosidosis causing mutation results in a change in therestriction sites in the gene and so there is a change in the patternwhen the digested fragments resulting from treatment with a particularrestriction enzyme are separated on a gel. The appearance and/ordisappearance of a particular sized fragment, indicated by a band on agel, is a simple way of detecting a mutant allele.

Clearly, any analysis of DNA potentially containing the disease causingmutation would be enhanced by the use in the test of an amplified amountof the particular DNA fragment around the mutation site. If the fragmentof interest can be amplified, small amounts of starting material may beemployed, for example, a few hair roots. As mentioned above, any of thein vitro amplification techniques known and described in the literaturemay be used. The polymerase chain reaction (PCR) and its modificationswill generally be the principal method of choice. PCR techniques requireprimer sequences which are complementary to DNA sequences upstream anddownstream of the disease causing mutation. These primers are used toamplify the DNA region that is located between them. The identificationof suitable oligonucleotide primers constitutes a further aspect of thepresent invention. Suitable primers may be designed with reference tothe cDNA and genomic sequences of the bovine LAMAN gene which areprovided according to the present invention. Thus, for example, primersmay be based on flanking sequences in either the cDNA or genomic DNA,which flank the site of the mutation, and suitable sequences may beselected from FIGS. 1 and 2 (SEQ ID Nos. 1 and 2) provided herein. Inthe case of the genomic bovine LAMAN sequence, primers may be based onboth flanking intron and exon sequences. For detection of the mutationin Angus and Angus-related breeds eg. Murray Grey cattle the followingprimer sequences are preferred:

5′CGCTGGACAC CCTAGCCTTA GGA 3′ (SEQ ID No.5) or

5′CGCAGGACACCCTAGCCTTAG-3′ (SEQ ID No.7) and

5′CCTTGCTATT GTTTTAAGCC TCTAAGTTT GTGGT 3′ (SEQ ID No.6)

The resulting amplified fragment is 296 base pairs and includes thenucleotide T₉₆₁ that is mutated in α-mannosidosis. In all cases,numbering of the nucleotides starts at the translation initiation codon.FIG. 4 shows the DNA sequence (SEQ ID No. 4) that is amplified duringthe PCR, indicates the primers (SEQ ID Nos. 5 and 6) and the mutatednucleotide. This T to C mutation introduces a further cleavage site forthe restriction endonuclease Mnl I.

In order to detect the mutation in Galloway cattle, the followingprimers are preferred:

5′GGGCTGCGCG TGTCCTCCAC AA 3′ (SEQ ID No. 8)and

5′CAGAAAATCG TGAGGGAACT GGTG 3′ (SEQ ID No. 9).

The following primer pair is also used to detect the mutation inGalloway cattle:

5′CGCAGGACACCCTAGCCTTAGGA 3′ (SEQ ID No.10)

5′CCTTGCTATTGTTTTAAGCCTCTAAGTTTGTGGT 3′ (SEQ ID No. 6).

This results in the amplification of a 1,700 base pair fragment or a 360base pair fragment which includes the disease causing mutation G₆₆₂ toA. This mutation removes a restriction site for the restrictionendonuclease Bsa HI which results in different size fragments ondigestion, easily detected on separation by gel electrophoresis.

Oligonucleotide primers may also be used in PCR or other in vitroamplification or primer extension-based methods of detecting mutations,eg. by PCR-SSP (PCR amplification with sequence specific primers, atechnique for detecting print mutations or allelic variations,traditionally used in tissue typing, see eg. Bunce et al., 1995, TissueAntigens, 45, 81-90) or the ARMS Technique of Zeneca for detectingvariant nucleotide sequences as described in EP-A-0332435.

Thus for example, primers may be designed wherein the terminal base ofthe primer at the 3′ end is designed to be complementary either to thenormal or mutant base, such that there is a terminal mismatch in theprimer, either with the mutant or with the normal allele; amplificationis much more efficient where there is a terminal match as opposed tomismatch, allowing the presence or absence of the mutation to bediscriminated, according to the amount of amplified product obtained.

The invention also extends to kits for the detection of disease causingmutations which comprise at least one oligonucleotide primer sequenceaccording to the invention. Such kits normally also contain additionalcomponents such as another oligonucleotide primer which hybridises tothe opposite strand of the target DNA and/or a restriction endonucleasesensitive to a bovine α-mannosidosis causing mutation The followingexamples are given by way of illustration only with reference to thefollowing figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence of bovine LAMAN cDNA (SEQ ID NO.1)and the deduced amino acid sequence (SEQ ID NO.2). The amino acidsequence is shown in single letter code below the nucleotide sequence.Both nucleotides and amino acids are numbered from the start of the openreading frame. The amino acid sequences colinear with N-terminal peptidedata are double underlined. Amino acid sequences that exhibitsimilarities with N-terminal amino acid sequences of human LAMANpeptides d and e are single underlined. The predicted polyadenylationsignal (ATTAAA) is marked in bold.

FIG. 2 shows the partial nucleotide sequence of bovine LAMAN genomic DNA(SEQ ID No. 3). The exons are written in upper case and the introns inlower case. Gaps in the intron sequence are indicated by askerisks (*)

FIG. 3 presents a scheme showing the different fragments produced whenamplified DNA encoding part of the LAMAN gene in Angus cattle is treatedwith the restriction enzyme Mnl I.

FIG. 4 shows a DNA sequence encompassing the T₉₆₁ mutation that isamplified by PCR (SEQ ID No. 4). The oligonucleotide primers are shownin small letters and the thymine nucleotide which is replaced by cystinein the mutated allele is surrounded by a box.

FIG. 5 is an Agarose gel stained with ethidium bromide showing theresults of electrophoresis following digestion of LAMAN DNA with Mnl I.

FIG. 6 illustrates SDS/PAGE and N-terminal amino acid sequencing. About1 mg/ml of purified LAMAN was subjected to SDS/PAGE either withoutpretreatment (left lane), prior heat denaturation (middle lane) or bothheat and reduction (right lane). The gel was stained with coomassieblue. For activity measurements the enzyme was applied to SDS/PAGEwithout pretreatment. After the run the gel was cut into several pieces,transferred into tubes containing PBS and following overnight diffusionthe enzyme activity was determined in the solution. For N-terminalsequencing the peptides were transferred to a PVDF-membrane byelectroblotting and sequenced by Edman degradation as described. Thepeptide sequences shown are SEQ ID Nos. 55-58. The 67 kDa band exhibitedmore than one amino acid in several of the cycles of Edman degradation(upper sequence, right side SEQ ID No. 55) indicating the presence ofmore than one N-terminal sequence within this band. Molecular massmarkers as described are indicated on the left side. The molecularmasses of the LAMAN peptides are shown on the right side. A model ofbovine LAMAN with arcs symbolizing disulphide bridges between peptidesis also shown.

FIGS. 7A, 7B and 7C show molecular shift analysis after endoglycosidaseH- and PNGase F-treatments. About 1 mg/ml of LAMAN was heat denaturedand treated with endo H and PNGase F. The glycosidase-treated sampleswere subjected to SDS/PAGE after reduction by β-mercaptoethanol andstained with coomassie blue (FIG. 7A) or blotted onto a PVDF-membraneand immunostained using peptide-specific antisera (FIG. 7B). Molecularmass standards (FIG. 7A, left side; FIG. 7B. right side) and molecularmasses of the LAMAN peptides (A, right side) are shown. In FIG. 7C,various types of oligosaccharide chains are: (▪) endo H-resistant; (□)endo H-sensitive; () mixture of endo H-resistant and sensitive. PartialN-glycosylation is symbolized by brackets and arcs symbolize disulphidebridges.

FIG. 8 shows the organisation of the LAMAN precursor. The six predictedcleavage sites of the LAMAN precursor are illustrated and the resultingproducts are designated as the signal peptide (SS), peptide a, peptideb, peptide c, pro, peptide d and peptide e. The amino acid position ateach cleavage is listed below the diagram. Potential N-glycosylationsites are indicated by black circles. The organisation of D. disc. LAMANprecursor is illustrated below the bovine LAMAN figure as a comparison.

FIGS. 9A and 9B show mutation detection in α-mannosidosis affected Angusand Galloway cattle. Partial sequence of the amplified bovine LAMAN cDNAfrom an α-mannosidosis affected Angus calf showing a T⁹⁶¹>C transitionresulting in Phe³²¹>Leu substitution is shown in (FIG. 9A). Partialsequence of amplified LAMAN cDNA from a Galloway heterozygous forα-mannosidosis showing a partial G⁶⁶²>A transition resulting inArg²²¹>His substitution is shown in (FIG. 9B).

FIG. 10 shows the sequences of the intron/exon boundaries of the bovineLAMAN gene (SEQ ID Nos. 59-104). The exon sequences are shown in uppercase letters, the intron sequences in lower case.

FIG. 11, Part A shows the genomic organisation of LAMAN gene betweenintron 2 and intron 7. Positions of the primers (mp30, mp262, mpi6F,mpi7R), Mnl I (M) and Bsa HI (B) restriction sites of the two ampliconsare indicated. Asterisks indicate the Bsa HI site lost due to the G662Amutation and the Mnl I sites introduced by the T961C mutation.

Part B, in the left gel: shows Bsa HI digestion of a 1700 bp PCR productamplified from Galloway animals using the primer combination mp30/mp262.Amplicons from normal animals are digested to four fragments (123 bp+380bp+420 bp+780 bp), while for affected three fragments are observed (123bp+380 bp+1200 bp). In carriers all fragments are present.

In the right gel: Mnl I digestion of 295 bp amplicaons from Angusanimals using primers mpi6F and mpi7R are shown. Digestion of ampliconsfrom normal results in fragments of 29 bp+266 bp, while amplicons fromaffected are digested to fragments of 29 bp+101 bp+165 bp. In carriersall fragments are observed.

EXAMPLE 1

Polymerase chain reaction (PCR) amplification of the sequence thatencompass the T₉₆₁→C (Phe₂₇₁→Leu) mutation

The method includes the amplification of a DNA fragment by PCR. Theamplified fragment is 296bp including the nucleotide Tg₆₁ that ismutated in α-mannosidosis. FIG. 4 shows the DNA sequence that isamplified by PCR. Oligonucleotide primers for amplification are called 1and 2. Sequences for the primers are respectively

5′CGCTGGACAC CCTAGCCTTA GGA 3′ (SEQ ID No.5) (1) and

5′CCTTGCTATT GTTTTAAGCC TCTAAGTTT GTGGT 3′ (SEQ ID No.6) (2).

The PCR amplification is typically carried out in a 100 μl volume with20 pmole of each primer, 100 ng genomic DNA, 250 μl of each DNTP, 2 UTaq DNA polymerase, 2 mM Mg²⁺ and 1×PCR buffer (10 mM HCl, pH 8.3containing 50 mM KCl). PCR conditions are: 95° C. for 5 minutes followedby 34 cycles where the temperature varies between 66° C. for 2 minutesand 94° C. for 1 minute. Finally the reaction mixture is incubated for 7minutes at 72° C.

Analysis of the PCR product

The PCR product is cut by the restriction enzyme Mnl I (P. Brinkley, etal., 1991, Gene, vol. 100, 2670-2682) at 37° C. for three hours. A Mnl Irestriction site was introduced in primer 2 (underlined in FIG. 4). Thissite fuctions as a positive control for the Mnl I enzyme activity. ThePCR product from normal cattle will be cut into fragments of 29 bp+267bp whereas affected cattle with the T₉₆₁→C (Phe₂₇₁→Leu) mutation willbut cut into DNA fragments of 29 bp+101 bp+166 bp. The carriers haveboth a normal and a mutated allele. Thus all the four DNA fragments willbe present after Mnl I cutting of the PCR products from carriers. Thefragments are separated by agarose gel electrophoresis. A 4% agarose gelwith 1×TBE (89 mM Tris-borate, pH 8.0 containing 2 mM EDTA) aselectrophoresis buffer is used. The DNA fragments are visualized usingethidium bromide staining and ultraviolet illumination (J. Sambrook, etal., 1989, In: Molecular Cloning: A Laboratory Manual). An example of anagarose gel electrophoresis is shown in FIG. 5.

EXAMPLE 2

Purification, cDNA sequencing and genomic organisation of bovinelysosomal α-mannosidase; characterisation of two mutations that causebovine α-mannosidosis

Enzyme assay

Lysomal α-mannosidase was assayed in 0.1 M acetate buffer, pH 4.5 and 4mM p-nitrophenyl α-D-mannopyranoside (SIGMA) at 37° C. Biochem. J., 71:318-323) or in 0.1 M acetate buffer, pH 3.7 and 4 mM4-methylumbelliferyl α-D-mannopyranoside (SIGMA). One unit of activityis defined as the amount of enzyme that liberates one μmol ofp-nitrophenol per min under the assay conditions.

Purification of α-mannosidase (summarized in Table 1 below)

TABLE 1 Purification of bovine kidney LAMAN Total Total SpecificPurification Protein activity activity Purification Yield step (mg)(units) (units/mg) (fold) (%) Crude extract 80000 1000 0.013 1 100 (10kg of kidney) Heat treatment 30000 900 0.03 2.3 90 (60° C., 30 min)Concanavalin 1100 720 0.65 50 72 A-sepharose Hydroxylapatite 230 600 2.6200 60 DEAE anion 10 150 15 1154 15 exchange Superdex 200 gel 5 100 201538 10 filtration

The first step was carried out at 4° C., while all the following stepswere performed at room temperature.

Step 1 Preparation of a crude extract

Ten kg of bovine (Norwegian red cattle) kidney that was obtained freshlyfrom a local slaughterhouse was cut into small pieces and homogenized in0.075 M acetic acid/0.15 M NaCl 1:2 (w:v) using a Waring blendor. Thehomogenate was centrifuged at 10.000 g for 10 min. To the supernatantwas added ammoniumsulfate to 35% saturation and stirred for at least 4 hand centrifuged at 10.000 g for 10 min. Ammoniumsulfate was then addedto 75% saturation and after stirring the solution was centrifuged asbefore. The resulting pellet was dissolved in a minimum amount of 0.05 Msodium phosphate buffer, pH 7.4/0.15 M NaCl (PBS). The final volume wastypically 41 and the solution was labelled “crude extract”.

Step 2 Heat treatment

Since bovine lysosomal α-mannosidase is stable at high temp. (Winchesteret al., 1976, Biochem. J., 157: 183-188) the crude extract was broughtto 60° C. and kept at that temperature for 20 min. The precipitate wasremoved by centrifugation at 10,000 g for 10 min.

Step 3 Concanavaline A-Sepharose

To the supernatant was added 50 ml concanavalin A (con A)-Sepharose(Pharmacia). The suspension was mixed for 2 h using a magnetic stirrer,and then run through a column. The resulting column of con A-Sepharose(2.5×10 cm) was washed with PBS. LAMAN was eluted by 200 ml PBScontaining 0.2 M α-methylmannoside.

Step 4 Hydroxylavatite chromatography

The eluate from the con A-Sepharose was applied to a 1.5×15 cm column ofhydroxylapatite (Biogel HTP, Bio-Rad) equilibrated with PBS. Elution wascarried out with 0.25 M sodium-phosphate, pH 7.4/0.15 M NaCl. The eluatewas dialysed against 4 1 or 0.02 M Tris/HCl, pH 7.6 with two shifts.

Step 5 O-Sepharose anion exchange chromatography

The dialysate was applied to a Q-Sepharose (Pharmacia) column (2×11 cm)equilibrated with 0.02 M Tris/HCl, pH 7.6 at a flow rate of 0.8 ml/min.LAMAN activity appeared in the run through and was concentrated throughan Amicon ultrafiltration unit fitted with a YM 30 membrane.

Step 6 Superdex 200 size exclusion chromatography

The concentrated sample was applied to Superdex 200 (Pharmacia) (1.5×60cm) equilibrated with PBS at a flow rate of 0.4 ml/min. The fractionscontaining LAMAN activity were collected and concentrated throughCentricon 30 microconcentrator (Amicon).

Aminoterminal sequencing

N-terminal amino acid sequencing of the LAMAN peptides were carried outas previously described (Tollersrud and Aronson (1989) BiochemicalJournal, 260 pp 101-108).

Generation of antibodies

Polyclonal antisera were obtained by immunization of male Chinchillarabbits with native LAMAN, the 67 kDa (peptide abc) and the 38 kDa(peptide d) peptide respectively according to standard protocols.Affinity purified antibodies against the 35 kDa peptide (peptide a) andthe 22 kDa peptide (peptide c) were prepared by incubatingpoly(vinylidine difluoride) (PVDF)-membrane strips (Immobilon™,Millipore), onto which the 35 kDa peptide or the 22 kDa peptide had beenblotted, with ⅕ diluted anti(67 kDa peptide )serum in 0.01 M Tris/HCl,pH 8.0/0.15 M NaCl/0.05% Tween 20 (TBST). The samples were kept 1 hourat room temp. and overnight at 4° C. The pieces of membrane were washedthree times with PBS and eluted with 0.2 M glycine, pH 2.8. The eluateswere immediately neutralized by ⅓ volume of 10×PBS.

Isolation and sequencing of laman cDNA

Degenerate oligonucleotide primers were designed to amplify LAMAN cDNAfrom a bovine kidney Lambda ZAP cDNA library (Stratagen). The forwardprimer aaMA1F: 5′ ATITACAAIACIGTICCIAAIGTIAAICC 3′ (SEQ ID No. 11)wasdeduced from amino acids 2-11 in the 35 kDa peptide (FIG. 6) with theambiguous amino acids in pos. 2 and 6 assigned Ile and Val respectively.The reverse primer abMA1R: 5′ ACIGCCATIGCITCATTIAGIGGIGC 3′ (SEQ ID No.12) was deduced from amino acids 5-13 of the 22 kDa peptide (FIG. 6). Toobtain bovine laman cDNA, DNA extracted from the library was subjectedto a two stage PCR. In the first round of 10 cycles the primerconcentrations were 10 fM and they were increased to 0.1 nM for afurther 30 cycles. For each cycle denaturation for 4 min at 94° C.followed by annealing at 43° C. for 3 min and extension at 70° C. for 3minutes. The resulting PCR product of about 1200 bp was cloned into theTA-cloning vector pCRII, as described by the manufactorer (Invitrogen)and then subcloned in both orientations into M13 mp18 by EcoRIdigestion, gel purification and ligation. The clone was sequenced withthe M13-40 universal primer and by primer walking. The cloned PCRfragment was used to screen 10⁶ plaques from the bovine kidney LambdaZAP cDNA library. Three independent clones were isolated, subcloned intoM13, and sequenced in both orientations. The 5′ end of the cDNAcontained an inverted repeat likely to form a stem-loop structure with astem size of 58 nts and a loop size of 148 nts. This inverted repeat wasprobably created by abberant cDNA synthesis during the libraryconstruction.

Isolation of a 5′ cDNA fragment

Genomic DNA isolated from bovine fibroblasts from an Angus bull (cc87/888) was digested with ApaI, diluted to 2 μg/ml and religated. Thecircularised DNA was subjected to PCR using the reverse primer mph266R5′AGAGAGGCGGGAGCGGTGG3′ (position 106-88) and the forward primer mp305′CAGAAAATCGTGAGGGAACTGGTG3′ (position 409-432) SEQ ID No. 14. After aninitial denaturation at 94° C. for 5 min, 7 cycles at 98° C. for 20 secand extension at 70° C. for 2 min were followed by 34 cycles at 98° C.and extension a 67° C. The 1500 bp amplicon product produced from thecircularized religated ApaI digest was cloned into pCRII and partiallysequenced. A part of this sequence was used as forward primer mp5UT1F5′GTGGCGGCGGCGGCTGCAGA3′ (position 4-23 SEQ ID No. 15) in combinationwith reverse primer mp262 5′GGGCTACGCGTGTCCTCCACAA3′ (position 836-857)SEQ ID No. 16 obtain an RT/PCR product of 800 bp that constituted a 5′part of the LAMAN cDNA.

Genomic organization

PCR with Taq DNA polymerase and with a mixture of Taq and Pwo DNApolymerases (Expand Long Template PCR System—Boehringer Mannheim) wasused to amplify fragments from genomic DNA isolated from a Charolaisbull. For each fragment annealing conditions were optimised to obtain asingle band evident upon electrophoresis in agarose. Length of eachamplified fragment were determined by comparison with molecular weightmarkers (Boehrienger Mannheim and Gibco BRL). Fragments amplified fromgenomic DNA were purified by PEG precipitation and sequenced with theprimers in the following Table.

Primers used for the determination of the genomic organisation (SEQ IDNos. 17-53)

Sequence of primer Primer#^(a) Location Position 5′-3′ EMAI 99 e-188-106 CCACCGCTCCCGCCTCTCT EMAI 146 i-1 −38/−19 ACTGACAGAGTGAGTGTGTGEMAI 88R e-2 224-206 TCATCATGTGTGTGAGGCA EMAI 185 e-3 317-338TCTCTTCCTTGCTGGCGAATCC EMAI 81 e-3 409-432 CAGAAAATCGTGAGGGAACTGGTG EMAI148 i-3/e-4 −7/440-451 GGAACAGGACGCCTAGAGT EMAI 83R e-4 534-513CGCAGTCTGAGTGTCATCTGG EMAI 84 e-5 694-714 AAGACGCTGCAGATGGAGCAGG EMAI149R e-5/i-5 765-766/+18 TCCACTCTACCACCTTACTG EMAI 82R e-6 857-836GGGCTACGCGTGTCCTCCACAA EMAI 142 e-6 836-855 TTGTGGAGGACACGCGTAGCCC EMAI100R e-7 990-971 GAACCACGTGTTGGCATTCT EMAI 109R i-7 +135/+107CCTTGCTATTGTTTTAAGCCTCTA  AGTTTG EMAI 85 e-7 1002-1024CAAGCTCATCCAGTTGGTCAATG EMAI 141 e-8 1046-1067 TCCGCGTCAATGTTCTCTACTCEMAI 80R e-9 1124-1102 TTCACTGACCAGCTGAGGTTGGC EMAI 139R e-11 1364-1344GAGGTACCACTGACTGCATCA EMAI 143 e-11 1342-1363 CATGATGCAGTCAGTGGTACCTEMAI 140 e-12 1482-1502 TTGTCGCAAGCTCAACATCAG EMAI 139R e-12 1531-1511TCTCTGCTGTCTGCGTGAGTG EMAI 89 e-14 1677-1699 TCAGGAGCTGCTTTTCTCAGCCTEMAI 144R e-14 1737-1718 GGAGACTGAGTAGATGCTGA EMAI 97R e-15 1892-1869TAGAAGGCTTGGCGAACAGGCAGC EMAI 91 e-16 1941-1959 GGTGCCTACATCTTCAGAC EMAI134R i-16 +79/+61 CCTCACAAGGCACATCACG EMAI 135 i-16 −46/−28GCTCAGGTGCACGCTTACA EMAI 92 e-18 2216-2194 CGGCCATTGCTGTCAGTGAGAA EMAI94 e-19 2286-2305 AAATTACTATCCAGTCAACA EMAI 133R i-19 +79/+60GGAGATGTCAAGAACAGGTG EMAI 90R e-21 2502-2480 CAGCACGAGGTGACGTCCTCGCAEMAI 95 e-21 2553-2574 GGAGGTCCTGGCCCCGCAGGT EMAI 128 e-22/i-222784-2787/+17 GACGGTGAGGATAGAGATGGA EMAI 96R e-23 2851-2830GGAGCTGGTTGGCCGCCAGCGT EMAI 93 e-23 2859-2879 CGCCTCCAGGCTCCAGTGGAC EMAI127R i-23 +133/+110 TCTGAACACCGACTTACTCCGACC EMAI 126R i-23 −70/−87CCTGAACACAGACATACC EMAI 86R e-24 3097-3072 GGCTTTCATTGGTGGTAGTAAGAG CA^(a)R at the end refers to reverse primer

Western blotting

Purified LAMAN (0.1 mg/ml) was subjected to SDS/PAGE on a Phast gelsystem (Pharmacia) using a gradient of 8-25% polyacrylamide. Afterelectrophoresis, the gel was overlayed with a PVDF membrane, andblotting was carried out by diffusion at 70° C. for 2 hours. Themembrane was blocked for 20 minutes with 2% (w/v) bovine serum albuminand incubated with {fraction (1/500)} diluted antiserum in TBST for 30minutes at room temperature. The immuno-complex was detected with asecondary antibody/alkaline phosphatase conjugate kit (Bio-Rad).

SDS/PAGE

SDS/PAGE was carried out with the Phast system from Pharmacia using an8-25% gradient of polyacrylamide or the mini gel apparatus from Bio-Radwith 10% polyacryamide in separating gel. For molecular mass analysisthe standards were (Pharmacia): α-lactalbumin (14.4 kDa), soyabeantrypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa), ovalbumin (43kDa), bovine serum albumin (67 kDa) and phosphorylase b (94 kDa).

Deglycosylation

10 μl LAMAN (1 mg/ml) was added SDS to a final concentration of 1% andkept in boiling water for 2 minutes. 90 μl 20 mM sodiumphosphate buffer,pH 7.2/50 mM-EDTA/0.5% Nonidet P-40 was added. After cooling 0.4 unitspeptide:N-glycanase F (PNGase F) or 2 units endoglycosidase H (endo H)(Boehringer Mannheim) was added and the solution incubated at 37° C.overnight. The deglycosylated enzyme was analyzed by SDS/PAGE.

Purification of LAMAN

The purification procedure is summarized in Table 1. The finalpreparation had a specific activity of 20 U/mg, which is in the sameorder as previous reports on purified LAMANS.

The purity was further assessed by SDS/PAGE. The enzyme remained activeafter SDS/PAGE and was detected at the point of application and thestacking/separating gel interface (FIG. 6). These bands probablycontained the native LAMAN complex that migrated slowly due to the lackof sufficient SDS-coating, as previously reported for two otherSDS-resistant enzymes, superoxide dismutase and glycosylasparaginase(Tollersrud and Aronson, 1989). Four faint lower molecular weight bandswere also detected (FIG. 6, left lane) that presumably were SDS-coatedpeptides resulting from partial denaturation of the native complex. Theincreased staining intensity of these bands after heat-denaturationconcomitant with the loss of the two activity containing bands confirmedthat they were dissociated peptides of the native LAMAN complex. Theabsence of bands that did not exhibit this correlation indicated thatthe enzyme was pure. Its isoelectric point was about 9.

Bovine LAMAN consists of 5 glycopeptides

Gel filtration on Superdex 200 indicate that the molecular mass ofnative LAMAN is approximately 250 kDa. FIG. 6 illustrates that there arefour peptides within LAMAN (center lane) and that breaking disulphidebonds fractionates the 67 kDa peptides to 35 kDa and 22 kDa fragments(right lane) leaving a putative 10 kDa fragment undetected. Withknowledge of the multiple N-terminal amino acid sequences of the 67 kDapeptide and of the 35 kDa and 22 kDa fragments (FIG. 6, right side) thededuced N-terminal sequence of the putative 10 kDa peptide would beNH₂XXXVNXXYST SEQ ID No. 54.

Panels A and B of FIG. 7 illustrates the effects of endo H and PNGase Ftreatments upon the various peptides. Peptides in panel A were detectedwith coomassie blue and those in panel B immunostained with peptidespecific antibodies. The peptides were designated a-e according to theirposition in the one chain precursor as illustrated in FIG. 8.

Antibodies against the 35 kDa peptide (peptide a) recognized a 38 kDaendo H-resistant form (FIG. 7B). However, following PNGase F treatmentonly a 35 kDa peptide was detected (FIG. 7B, left lane) indicating thatthis 38 kDa peptide is a glycosylated variant of the 35 kDa peptide andthat the sugar chain is of complex type.

Antibodies against the 22 kDa peptide (peptide c) recognized 3additional peptides of 70 kDa, 35 kDa and 33 kDa (FIG. 7B, right blot)that were present in low concentrations as they were not detected incoomassie blue stained gel. Given molecular masses it is probable thatthe 70 kDa peptide is a one chain precursor of the 67 kDa bandconsisting of peptides a, b and c. Similarly, the 35 kDa and 33 kDabands are most likely precursors of the 22 kDa peptic and the putative10 kDa peptide (peptide b). After both endo H and PNGase F treatmentsthe 35 kDa and 33 kDa peptides detected with the anti (22 kDapeptide)body, merged to a 29 kDa band (FIG. 7B, right blot) indicatingthat their sugar chains are not of the complex type. As both PNGase Fand endo H treatments transformed the 22 kDa to an 18 kDa peptide (FIGS.7A and 7B) the 29 kDa unglycosylated band was composed of an 11 Dapeptide in addition to the 18 kDa peptide. This 11 kDa peptide isprobably the putative 10 kDa peptide that was not observed on SDS/PAGE(FIG. 6). It is probable then that the 33 kDa and 35 kDa peptides thatcrossreacted with anti (22 kDa peptide) body contain an unglycosylatedand a glycosylated form of this 11 kDa peptide respectively.

The 38 kDa peptide (peptide d) was catabolised to a 35 kDa band afterendo H-treatment and to 29 kDa after PNGase F-treatment (FIG. 7, panelB, left blot) suggesting that the peptide had one sugar chain of highmannose and two of the complex type. After PNGase F-treatment the 15 kDapeptide (peptide e) merged with the 13 kDa peptide suggesting that theformer is a N-glycosylated variant of the latter. However, some 15 kDapeptide persisted after endo H-treatment. Partial N-glycosylation oflysosomal enzymes has been reported previously. A model of the peptidesand their N-glycosylation pattern is shown (FIG. 7C).

The LAMAN peptides originate from a single gene

The aminoterminal amino acid sequences of the 35/38 kDa (peptide a) and22 kDa (peptide c) peptides exhibited 40% identity with two internalsequences within D. disc. LAMAN (Schatzle et al., 1992) separated by 376amino acids. With degenerate oligonucleotides deduced from these aminoacid sequences as primers a 1200 bp fragment was amplified from a bovinekidney lambda ZAP cDNA library. The amino acid sequence deduced from theamplified fragment was 49% identical with D. disc. LAMAN and containedan amino acid sequence similar to the aminoterminus of peptide b. The1200 bp amplified product was used as a probe to screen the bovinekidney λ ZAP library. Sequencing the 3300 bp insert in a positive clonerevealed an open reading frame. The deduced amino acid sequencecontained the aminotermini of each of the LAMAN peptides and a 136 bpuntranslated region with a poly A signal 13 bp upstream of a poly A tail(FIG. 1). The 5′ end of the cDNA had an inverted loop of 200 bp that wasconsidered likely to be an artifact of the library construction. Primersbased on the sequence of a circularised genomic fragment from anupstream region of the laman gene were used in RT/PCR to obtain the 5′end of the cDNA. Two in frame ATGs were detected that were 150 and 288nucleotides respectively upstream of the first nucleotide encoding theN-terminus of peptide a. Two forward primers were constructed thatcorresponded to sequences immediately upstream of each ATG codon andwere used in combination with a reverse primer mp262 for RT/PCR asdetailed in the experimental section. Only the primer immediatelyupstream of the second ATG resulted in a PCR product of the expectedsize of 800 bp, while both combinations resulted in the expected size ofabout 2000 bp when PCR was carried out using genomic DNA as template(not shown). Thus the second ATG is probably the translation initiationsite. This region conformed with the Kozak concensus sequence (Kozak,1991). The DNA sequences from the RT/PCR product and the λ ZAP cDNAclone were combined to yield a full length cDNA sequence encoding theentire bovine LAMAN polypeptide (FIG. 1).

A comparison of the molecular masses of the deglycosylated peptidesdetermined by SDS/PAGE and by calculation from the deduced amino acidsequence revealed that they were similar (Table 2) except for peptide cthat was 21.3 kDa according to the deduced cDNA sequence, while the Mrof the deglycosylated peptide was 18 kDa according to SDS/PAGE.

TABLE 2 Comparison between molecular masses as judged by SDS/PAGE andcalculated from the deduced cDNA sequence Glyco- M_(r) (kDa) M_(r) (kDa)on M_(r) (kDa) M_(r)-shift sylation on SDS/PAGE calc. from (kDa) sitefrom SDS/ after deduced after deduced Peptide PAGE PNGase F cDNA seq.PNGase F cDNA a 35/38 35 34.5 0/3 1 b 11/13 11 9.6 0/2 1 c 22 18 17.4 41 d 38 29 28.4 9 4 e 13/15 13 14.1 0/2 1 total 119/126 106 104 13/20 8

In human LAMAN there is a cleavage site corresponding to bovine LAMANposition 591 that is 31 amino acids N-terminally to the cleavage sitethat generated the N-terminus of peptide d. Assuming that this humancleavage site is conserved in bovine LAMAN the Mr of peptide c from thededuced cDNA sequence would be in agreement with its relative migrationon SDS/PAGE after deglycosylation (Table 2). A model of the cleavagepattern and N-glycosylation sites is illustrated (FIG. 8). In this modelthe peptides are named a to e according to their original positionrelative to the N-terminus of the LAMAN one chain precursor.

Alpha-mannosidosis among Angus cattle

The α-mannosidase activity in a liver extract from affected Angus cattlewas 0.3% of normal as previously reported by Healy et al., 1990, Res.Vet. Sci., 49: 82-84. Half of this activity was immunoprecipitatedindicating that LAMAN was expressed at 0.15% of normal activity. Theresidual activity that did not immunoprecipitate was also found in anormal liver extract. An acid α-mannosidase activity from humanAlpha-mannosidosis fibroblasts with similar properties has previouslybeen reported indicating that this is a different gene product. Westernblot analysis of fibroblast extracts showed a decrease of expression ofpeptide abc.

LAMAN fibroblast cDNA from normal and affected Angus cattle werecompared by direct sequencing of PCR products. A single nucleotidetransversion, T⁹⁶¹>C, was discovered that was predicted to result inphe³²¹>leu substitution (FIG. 9). Sequence alignment studies withα-mannosidase sequences from other species (eg. yeast, rat, D. disc,cow, human, mouse, pig and Drosophila) have shown that Phe³²¹ isconserved within the class 2 α-mannosidase family indicating that itserves an important physiological function. Recently we discovered thatthe T⁹⁶¹>C transversion was conserved in both alleles of three affectedAngus cattle, in one of the alleles of twelve carriers and in no allelesof fifty-eight normal Angus cattle.

Alpha-mannosidosis among Galloway cattle

The α-mannosidase activity in a liver extract from Alpha-mannosidosisaffected Galloway cattle was 1.8% of normal. A similar activity wasdetected in the liver extract from an affected Angus x Gallowaycrossbreed. About 90% of these activities were immunoprecipitatedindicating that they resulted predominantly from the expression ofLAMAN. The temperature stability of LAMAN from affected Galloway cattlewas different from the LAMAN stability from normal cattle and affectedAngus cattle. LAMAN from affected Galloway was heat denatured in twophases, 80% being denatured with T_(½) of 60° C. and 20% denatured withT_(½) of 83° C. Both normal LAMAN and LAMAN from affected Angus was heatdenatured in one phase with T_(½) of 70° C.

Comparison of the sequences of normal LAMAN cDNA and fibroblast cDNAfrom a carrier of Galloway breed revealed a difference in a singleposition. A single nucleotide transversion, G⁶⁶²>A was discovered thatwas predicted to result in arg²²¹>his substitution. In a Gallowaycarrier there are two bases in position 662 because of the presence of anormal allele (G662) and a mutated allele (A662) (FIG. 9). In thisposition only arg or lys appear within the class 2 α-mannosidase familyindicating that these closely related side chains are physiologicallyimportant at this site. The G⁶⁶²>A transition was recently discovered inboth alleles of two affected Galloway cattle, in one of the alleles ofseven carriers and in no alleles of twenty-nine normal Galloway cattle.

Organisation of the LAMAN gene

The intron-exon boundaries were characterised by sequencing of PCRproducts from bovine genomic DNA using as primers oligonucleotidesequences from the bovine cDNA or intronic sequences that was determinedfrom the PCR products. The LAMAN gene spanned approximately 16 kb andconsisted of 24 exons. Exon 1 encoded the signal peptide and 4 aminoacids of peptide a. Exon 24 encoded 36 amino acids at thecarboxyterminal end of peptide e. All intron-exon junctions fitted theGT/AG consensus rule (FIG. 10).

Summary

We have purified to homogeneity and characterised lysosomalα-mannosidase (LAMAN) from bovine kidneys. Its cDNA containing thecomplete open reading frame of 2997 bp was sequenced. The mature enzymeconsisted of five different peptides. Four of these peptides wereaminoterminally sequenced and found to be identical to internalsequences within the deduced cDNA sequence. The peptide that was notsequenced, the 13/15 kDa peptide was identical in molecular mass to ahuman LAMAN peptide. The N-terminal amino acid sequence of this humanpeptide was more than 60% identical to pos. 872-889 of the deducedbovine LAMAN amino acid sequence indicating that position 872 is apotential cleavage site. Such a cleavage would generate a peptide of theexpected size of 14 kDa. Thus, bovine, LAMAN was probably synthesized asa one chain precursor that was processed into five peptides.

The amino acid sequence exhibited 83% identity with the deduced aminoacid sequence of a putative human retina LAMAN cDNA (Nebes, V. L. andSchmidt, M. C., 1994, Biochem. Biophys. Res. Comm., 200: 239-245) exceptfor regions that in the bovine LAMAN sequence are located in pos.510-550, 571-588 and 865-898. A comparison with the deduced amino acidsequence of a human placenta LAMAN cDNA revealed a high degree ofsimilarity with the bovine sequence throughout the entire sequence.

To distinguish the peptides within the LAMAN complex we suggest to namethem in alphabetic order according to their original position relativeto the N-terminus of the one chain precursor. Thus, the 35/38 kDapeptide was named peptide a, the 11/13 kDa peptide was peptide b, the 22kDa peptide was peptide c, the 38 kDa peptide was peptide d and the13/15 kDa peptide was peptide e (Table 2). This peptide pattern hasnever before been reported for any LAMAN.

Prior to reduction there were three peptides of 67 kDa, 38 kDa and 13/15kDa of which the 67 kDa peptide contained peptide a, peptide b andpeptide c joined by disulfide bridges.

In addition to exon 1 that encoded the signal peptide there were 23exons within a 16 kb region. The exon/intron boundaries were identicalto those in human LAMAN but the intron sizes varied. The elucidation ofthe exon/intron organisation is important for the construction ofDNA-based mutation detection systems for bovine Alpha-mannosidosis andfor the study of exon/intron organisation of LAMAN in other species.

One of the aims of this study was to determine if the variation of theclinical expressions between alpha-mannosidosis affected cattle ofGalloway and Angus breeds could be explained by a different genotype.Affected cattle from both breeds expressed acid α-mannosidase activitiesthat were precipitated by the antersium. This indicated that themutations did not result in a major disruption of the LAMAN polypeptidechain. Thus, not unexpectedly the mutations G662A and T961C werenucleotide substitutions that resulted in the single amino acid changesR221H and F321L. The mutated enzyme from Angus appeared to be similarlyheat stable as normal LAMAN, but more heat stable than the mutatedenzyme from Galloway cattle confirming that the disorders in Angus andGalloway were caused by different mutations.

The mutation in Angus apparently resulted in the reduced expression ofLAMAN polypeptide, while the temperature stability and specific activityappeared unchanged. It is possible that the mutation causeddestabilization of a folding intermediate, resulting in the majority ofthe precursor being misfolded and prematurely degraded. The mutation inGalloway, however, caused a change of the temperature stabilityindicating that the conformation of the mature enzyme had been changed.The biphasic temperature denaturation event suggested that the mutatedenzyme exists in different conformational isoforms. While the Angusmutation apparently resulted in the substitution of a highly conservedphenylalanine for a leucine within a region containing few conservedamino acids among class 2 α-mannosidases the mutation in Gallowayresulted in the substitution of arginine for histidine within a highlyconserved region. Possibly, this arginine that is replaced by lysine inER and yeast α-mannosidases functions close to the active site, and thatits substitution results in local conformational changes with highimpact on the enzymatic activity. Such a substitution might also alterthe substrate specificity with a larger decrease of catalytic activitytowards naturally occuring oligosaccharides than against the simple,artificial substrate used in this study. This may explain the apparent10× higher LAMAN activity in affected Galloway than Angus despite themore severe clinical expression of the disease in Galloway cattle.

The differences of the enzyme properties combined with the variation ofthe clinical expression between the breeds clearly indicated thatmolecular heterogeneity exists for α-mannosidosis between the twobreeds. The carrier Galloway cow we examined lacked the T961>Ctransition for which the affected Angus was homozygous. Similarly theaffected Angus calf lacked the G662>C transition present in one of thealleles of the Galloway carrier. The T961>C mutation was found in eachof 8 affected Angus, in 12 heterozygous but not in 100 Angus classifiedas non-carriers on the basis of α-mannosidase activity in blood.Similarly the G662>C transition was found on both alleles in 4 affectedGalloway calves, 10 putative carriers were heterozygous for thismutation and the mutation was absent from 20 Galloways classified asnon-carriers on the basis of enzyme activity in blood. Since these twomutations were the only ones detected by sequencing the complete cDNAsand the corresponding amino acid substitutions involved amino acids thatwere conserved among class 2 α-mannosidases, we have concluded that theyare the disease causing mutations. Interestingly both mutations resultedin the expression of a small, but significant amount of LAMAN-activity.Although it is not known what effect this activity has on the clinicalexpression its presence in both affected breeds could indicate that someenzyme activity is necessary for the prenatal development. Studies ofα-mannosidosis causing mutations in human will indicate whetherexpression of enzyme activity is common among α-mannosidosis affectedindividuals. The elucidation of the disease causing mutations in cattlehave made possible an improved diagnostic tool for detecting carriers ofbovine α-mannosidosis.

EXAMPLE 3

Screening for the G662A and the T961C mutations by PCR based restrictionfragment length polymorphism (RFLP) analysis

Methods

The G662A and the T961C mutations are in exons 5 and 7 of the LAMANgene, (FIG. 11A). To Screen for the G662A mutation, a 1700 bp fragmentincluding exon 5 was amplified by PCR in 100 μl containing 100 ng ofgenomic DNA, 20 pmol of each primer

mp262 (5′-GGGCTGCGCGTGTCCTCCACAA-3′) (SEQ ID No.8) and

mp30 (5′-CAGAAAATCGTGAGGGAACTGGTG-3) (SEQ ID No.9),

100 μM dNTP's, 2U Taq DNA polymerase (Gibco-BRL), 1×PCR buffer (50 mMKCl, 10 mM Tris-HCl, pH 8.3) and 2 mM of Mg²⁺. The reaction was cycled34 times as follows: 66° C. for 3 minutes and 94° C. for 1 minute.Initial denaturation was 95° C. for 5 minutes and final extension was 7minutes at 72° C. Twenty μl of the PCR products were digested in the PCRbuffer overnight at 37° C. by 2U of Bsa HI.

The T961C mutation was detected by PCR amplification using the primercombination

mpi6F (5′-CGCAGGACACCCTAGCCTTAG-3′) (SEQ ID No. 7) and

mpi7R (5′CCTTGCTATTGTTTTAAGCCTCTAAGTTTGTGGT-3′)(SEQ ID No.6).

PCR conditions were as above. The resulting 290 bp PCR products includedexon 7 and were digested in the PCR buffer overnight by 2U Mnl I. A MnlI restriction site were introduced to the primer mpi7R to serve as apositive control for digestion. The digests were separated on a 2%agarose gel and visualized by EtBr staining (FIG. 11B).

Results

The G662A mutation removes a Bsa HI restriction site while the T961Cmutation introduces a Mnl I site. Hence, both mutations are easilydetected by PCR based RFLP on DNA extracted from blood. The tests werealso found to work on DNA from semen and hair roots. The assays wereused to screen for the mutations in animals whose α-mannosidosisgeneotype previously had been determined by measuring the LAMAN activityby the granulocyte or plasma test. The affected Angus cattle werehomozygous, and all carrier Angus, Red Angus and Murray Grey cattle wereheterozygous for the T961C mutation. No normal animals carried the T961Cmutation. The animals were from unrelated herds from different parts ofAustralia, except for 7 Red Angus of which 4 were carriers of T961C,providing evidence that the T961C mutation is present in North AmericanAngus breeds. All affected Galloways were homozygous, and the carrierGalloways were heterozygous for the G662A mutation. No normal animalscarried the G662A mutation.

104 3147 base pairs nucleic acid single linear cDNA NO NO unknown CDS15..3011 polyA_signal 3115..3120 1 GCGGCTGCAG AGCC ATG GTT GGT GAC GCGCGG CCT TCA GGG GTT CGC GCT 50 Met Val Gly Asp Ala Arg Pro Ser Gly ValArg Ala 1 5 10 GGC GGC TGC CGG GGC GCG GTA GGA TCC CGG ACG AGC TCC CGCGCG CTG 98 Gly Gly Cys Arg Gly Ala Val Gly Ser Arg Thr Ser Ser Arg AlaLeu 15 20 25 CGG CCA CCG CTC CCG CCT CTC TCC TCC CTC TTC GTG TTG TTC CTAGCG 146 Arg Pro Pro Leu Pro Pro Leu Ser Ser Leu Phe Val Leu Phe Leu Ala30 35 40 GCG CCC TGC GCT TGG GCG GCG GGA TAC AAG ACA TGC CCG AAG GTG AAG194 Ala Pro Cys Ala Trp Ala Ala Gly Tyr Lys Thr Cys Pro Lys Val Lys 4550 55 60 CCG GAC ATG CTG AAT GTA CAC CTG GTG CCT CAC ACA CAT GAT GAT GTA242 Pro Asp Met Leu Asn Val His Leu Val Pro His Thr His Asp Asp Val 6570 75 GGC TGG CTC AAG ACG GTG GAC CAG TAC TTC TAT GGC ATC TAC AAT AAC290 Gly Trp Leu Lys Thr Val Asp Gln Tyr Phe Tyr Gly Ile Tyr Asn Asn 8085 90 ATC CAG CCG GCG GGT GTA CAG TAC ATC CTA GAC TCC GTC ATC TCT TCC338 Ile Gln Pro Ala Gly Val Gln Tyr Ile Leu Asp Ser Val Ile Ser Ser 95100 105 TTG CTG GCG AAT CCC ACC CGC CGC TTC ATC TAT GTG GAA ATC GCC TTC386 Leu Leu Ala Asn Pro Thr Arg Arg Phe Ile Tyr Val Glu Ile Ala Phe 110115 120 TTC TCG CGT TGG TGG CGC CAG CAG ACA AAT GCA ACA CAG AAA ATC GTG434 Phe Ser Arg Trp Trp Arg Gln Gln Thr Asn Ala Thr Gln Lys Ile Val 125130 135 140 AGG GAA CTG GTG CGC CAG GGA CGC CTA GAG TTC GCC AAC GGT GGCTGG 482 Arg Glu Leu Val Arg Gln Gly Arg Leu Glu Phe Ala Asn Gly Gly Trp145 150 155 GTG ATG AAC GAT GAG GCG ACC ACC CAC TAC GGA GCC ATC ATT GACCAG 530 Val Met Asn Asp Glu Ala Thr Thr His Tyr Gly Ala Ile Ile Asp Gln160 165 170 ATG ACA CTC AGA CTG CGC TTC CTG GAG GAG ACG TTC GGC AGC GACGGG 578 Met Thr Leu Arg Leu Arg Phe Leu Glu Glu Thr Phe Gly Ser Asp Gly175 180 185 CGC CCC CGT GTG GCC TGG CAC ATC GAC CCA TTC GGC CAC TCT CGGGAG 626 Arg Pro Arg Val Ala Trp His Ile Asp Pro Phe Gly His Ser Arg Glu190 195 200 CAA GCT TCA CTG TTC GCG CAG ATG GGT TTT GAC GGC TTC TTC TTTGGA 674 Gln Ala Ser Leu Phe Ala Gln Met Gly Phe Asp Gly Phe Phe Phe Gly205 210 215 220 CGC CTG GAT TAT CAA GAC AAG AAG GTG CGG AAA AAG ACG CTGCAG ATG 722 Arg Leu Asp Tyr Gln Asp Lys Lys Val Arg Lys Lys Thr Leu GlnMet 225 230 235 GAG CAG GTG TGG CGG GCC AGC ACC AGC CTG AAA CCT CCC ACTGCC GAC 770 Glu Gln Val Trp Arg Ala Ser Thr Ser Leu Lys Pro Pro Thr AlaAsp 240 245 250 CTC TTC ACC AGT GTG CTC CCC AAC ATG TAC AAC CCG CCG GAAGGT CTG 818 Leu Phe Thr Ser Val Leu Pro Asn Met Tyr Asn Pro Pro Glu GlyLeu 255 260 265 TGC TGG GAC ATG CTG TGT GCC GAC AAG CCG GTT GTG GAG GACACG CGT 866 Cys Trp Asp Met Leu Cys Ala Asp Lys Pro Val Val Glu Asp ThrArg 270 275 280 AGC CCA GAG TAC AAC GCA AAA GAG CTG GTC CGT TAC TTC CTGAAG TTG 914 Ser Pro Glu Tyr Asn Ala Lys Glu Leu Val Arg Tyr Phe Leu LysLeu 285 290 295 300 GCC ACT GAC CAG GGT AAG CTC TAC CGC ACC AAA CAC ACTGTG ATG ACC 962 Ala Thr Asp Gln Gly Lys Leu Tyr Arg Thr Lys His Thr ValMet Thr 305 310 315 ATG GGC TCA GAC TTC CAG TAC GAG AAT GCC AAC ACG TGGTTC AAA AAT 1010 Met Gly Ser Asp Phe Gln Tyr Glu Asn Ala Asn Thr Trp PheLys Asn 320 325 330 CTT GAC AAG CTC ATC CAG TTG GTC AAT GCC CAG CAA CGGGCC AAC GGG 1058 Leu Asp Lys Leu Ile Gln Leu Val Asn Ala Gln Gln Arg AlaAsn Gly 335 340 345 ATC CGC GTC AAT GTT CTC TAC TCT ACC TCG GCC TGT TACCTC TGG GAG 1106 Ile Arg Val Asn Val Leu Tyr Ser Thr Ser Ala Cys Tyr LeuTrp Glu 350 355 360 CTG AAC AAG GCC AAC CTC AGC TGG TCA GTG AAA AAG GATGAC TTC TTC 1154 Leu Asn Lys Ala Asn Leu Ser Trp Ser Val Lys Lys Asp AspPhe Phe 365 370 375 380 CCC TAT GCT GAT GGC CCC TAC ATG TTC TGG ACC GGTTAC TTT TCC AGC 1202 Pro Tyr Ala Asp Gly Pro Tyr Met Phe Trp Thr Gly TyrPhe Ser Ser 385 390 395 CGG CCT GCC CTC AAA CGC TAC GAG CGT CTC AGC TACAAT TTC CTG CAG 1250 Arg Pro Ala Leu Lys Arg Tyr Glu Arg Leu Ser Tyr AsnPhe Leu Gln 400 405 410 GTG TGC AAC CAG CTG GAG GCG CTG GCG GGT CCG GCAGCC AAC GTG GGA 1298 Val Cys Asn Gln Leu Glu Ala Leu Ala Gly Pro Ala AlaAsn Val Gly 415 420 425 CCC TAT GGC TCC GGG GAC AGT GCA CCC CTC AAT GAGGCG ATG GCC GTG 1346 Pro Tyr Gly Ser Gly Asp Ser Ala Pro Leu Asn Glu AlaMet Ala Val 430 435 440 CTC CAG CAC CAT GAT GCA GTC AGT GGT ACC TCC CGGCAG CAC GTG GCT 1394 Leu Gln His His Asp Ala Val Ser Gly Thr Ser Arg GlnHis Val Ala 445 450 455 460 AAC GAC TAT GCC CGC CAA CTT TCA GAA GGC TGGAGG CCT TGC GAG GTT 1442 Asn Asp Tyr Ala Arg Gln Leu Ser Glu Gly Trp ArgPro Cys Glu Val 465 470 475 CTC ATG AGC AAT GCG CTG GCG CAT CTC AGC GGCTTA AAG GAG GAC TTC 1490 Leu Met Ser Asn Ala Leu Ala His Leu Ser Gly LeuLys Glu Asp Phe 480 485 490 GCC TTT TGT CGC AAG CTC AAC ATC AGC ATT TGTCCA CTC ACG CAG ACA 1538 Ala Phe Cys Arg Lys Leu Asn Ile Ser Ile Cys ProLeu Thr Gln Thr 495 500 505 GCA GAG AGA TTC CAG GTG ATC GTT TAT AAC CCCCTG GGG CGG AAA GTG 1586 Ala Glu Arg Phe Gln Val Ile Val Tyr Asn Pro LeuGly Arg Lys Val 510 515 520 GAC TGG ATG GTG CGG CTG CCT GTC AGC AAA CACGTT TAC CTC GTG AAG 1634 Asp Trp Met Val Arg Leu Pro Val Ser Lys His ValTyr Leu Val Lys 525 530 535 540 GAC CCC GGT GGC AAA ATT GTG CCC AGC GATGTG GTG ACC ATT CCC AGT 1682 Asp Pro Gly Gly Lys Ile Val Pro Ser Asp ValVal Thr Ile Pro Ser 545 550 555 TCA GAC AGT CAG GAG CTG CTT TTC TCA GCCTTA GTG CCT GCC GTG GGC 1730 Ser Asp Ser Gln Glu Leu Leu Phe Ser Ala LeuVal Pro Ala Val Gly 560 565 570 TTC AGC ATC TAC TCA GTC TCC CAG ATG CCTAAC CAA AGA CCC CAG AAG 1778 Phe Ser Ile Tyr Ser Val Ser Gln Met Pro AsnGln Arg Pro Gln Lys 575 580 585 TCC TGG TCC CGT GAC TTG GTC ATC CAG AATGAG TAC CTC CGG GCT AGG 1826 Ser Trp Ser Arg Asp Leu Val Ile Gln Asn GluTyr Leu Arg Ala Arg 590 595 600 TTT GAC CCT AAC ACA GGG CTC TTG ATG GAGTTG GAG AAC CTG GAG CAG 1874 Phe Asp Pro Asn Thr Gly Leu Leu Met Glu LeuGlu Asn Leu Glu Gln 605 610 615 620 AAT CTC TTG CTG CCT GTT CGC CAA GCCTTC TAC TGG TAC AAC GCC AGT 1922 Asn Leu Leu Leu Pro Val Arg Gln Ala PheTyr Trp Tyr Asn Ala Ser 625 630 635 ACA GGT AAC AAC CTA AGC TCC CAG GCCTCC GGT GCC TAC ATC TTC AGA 1970 Thr Gly Asn Asn Leu Ser Ser Gln Ala SerGly Ala Tyr Ile Phe Arg 640 645 650 CCC AAC CAG AAC AAA CCA CTG TTC GTGAGC CAC TGG GCT CAG ACC CAC 2018 Pro Asn Gln Asn Lys Pro Leu Phe Val SerHis Trp Ala Gln Thr His 655 660 665 CTT GTG AAG GCG TCC TTG GTG CAG GAAGTA CAC CAG AAC TTC TCA GCC 2066 Leu Val Lys Ala Ser Leu Val Gln Glu ValHis Gln Asn Phe Ser Ala 670 675 680 TGG TGT TCC CAG GTG GTT CGC CTG TATCCC AGA CAA CGG CAC CTG GAG 2114 Trp Cys Ser Gln Val Val Arg Leu Tyr ProArg Gln Arg His Leu Glu 685 690 695 700 CTA GAG TGG ACA GTG GGG CCA ATACCT GTG GGA GAC GGC TGG GGG AAG 2162 Leu Glu Trp Thr Val Gly Pro Ile ProVal Gly Asp Gly Trp Gly Lys 705 710 715 GAG GTC ATC AGT CGC TTT GAC ACTGCA TTG GCG ACA CGC GGA CTC TTC 2210 Glu Val Ile Ser Arg Phe Asp Thr AlaLeu Ala Thr Arg Gly Leu Phe 720 725 730 TAC ACT GAC AGC AAT GGC CGG GAGATC CTG GAG AGG AGG CGG AAT TAT 2258 Tyr Thr Asp Ser Asn Gly Arg Glu IleLeu Glu Arg Arg Arg Asn Tyr 735 740 745 AGA CCT ACC TGG AAG CTG AAC CAGACT GAA CCC GTG GCT GGA AAT TAC 2306 Arg Pro Thr Trp Lys Leu Asn Gln ThrGlu Pro Val Ala Gly Asn Tyr 750 755 760 TAT CCA GTC AAC AGC CGC ATT TACATC ACG GAT GGG AAC ATG CAG CTG 2354 Tyr Pro Val Asn Ser Arg Ile Tyr IleThr Asp Gly Asn Met Gln Leu 765 770 775 780 ACT GTG CTC ACT GAC CGG TCCCAG GGG GGC AGT AGC CTG AGA GAT GGC 2402 Thr Val Leu Thr Asp Arg Ser GlnGly Gly Ser Ser Leu Arg Asp Gly 785 790 795 TCC TTG GAA CTC ATG GTG CACCGA AGG CTG CTG AAG GAC GAT GCA CGC 2450 Ser Leu Glu Leu Met Val His ArgArg Leu Leu Lys Asp Asp Ala Arg 800 805 810 GGA GTT GGG GAG CCG CTG AACAAG GAG GGG TCG GGG CTT TGG GTG CGA 2498 Gly Val Gly Glu Pro Leu Asn LysGlu Gly Ser Gly Leu Trp Val Arg 815 820 825 GGA CGT CAC CTC GTG CTG TTGGAT AAG AAG GAG ACT GCG GCC GCC AGG 2546 Gly Arg His Leu Val Leu Leu AspLys Lys Glu Thr Ala Ala Ala Arg 830 835 840 CAC CGG TTA CAG GCG GAG ATGGAG GTC CTG GCC CCG CAG GTG GTG CTG 2594 His Arg Leu Gln Ala Glu Met GluVal Leu Ala Pro Gln Val Val Leu 845 850 855 860 GCT CAA GGT GGC GGC GCGCGG TAT CGC CTC GAG AAA GCC CCA CGC ACG 2642 Ala Gln Gly Gly Gly Ala ArgTyr Arg Leu Glu Lys Ala Pro Arg Thr 865 870 875 CAG TTC TCT GGG CTC CGCCGC GAG CTG CCA CCC TCG GTA CGT CTG CTC 2690 Gln Phe Ser Gly Leu Arg ArgGlu Leu Pro Pro Ser Val Arg Leu Leu 880 885 890 ACA TTG GCC CGC TGG GGCCCG GAG ACA CTG CTG CTG CGC TTA GAG CAC 2738 Thr Leu Ala Arg Trp Gly ProGlu Thr Leu Leu Leu Arg Leu Glu His 895 900 905 CAG TTC GCC GTA GGG GAGGAC TCG GGC CGG AAC TTG AGC TCC CCG GTG 2786 Gln Phe Ala Val Gly Glu AspSer Gly Arg Asn Leu Ser Ser Pro Val 910 915 920 ACC CTG GAC TTG ACG AACTTG TTT TCC GCC TTC ACC ATC ACC AAC CTG 2834 Thr Leu Asp Leu Thr Asn LeuPhe Ser Ala Phe Thr Ile Thr Asn Leu 925 930 935 940 CGG GAG ACC ACG CTGGCG GCC AAC CAG CTC CTG GCC TAC GCC TCC AGG 2882 Arg Glu Thr Thr Leu AlaAla Asn Gln Leu Leu Ala Tyr Ala Ser Arg 945 950 955 CTC CAG TGG ACG ACGGAC ACG GGC CCC ACA CCC CAT CCT TCT CCT TCC 2930 Leu Gln Trp Thr Thr AspThr Gly Pro Thr Pro His Pro Ser Pro Ser 960 965 970 CGT CCG GTG TCC GCCACC ATC ACG CTG CAG CCC ATG GAA ATC CGT ACC 2978 Arg Pro Val Ser Ala ThrIle Thr Leu Gln Pro Met Glu Ile Arg Thr 975 980 985 TTC TTG GCT TCG GTCCAA TGG GAA GAG GAC GGC TAGACCCACT GGATACAAGA 3031 Phe Leu Ala Ser ValGln Trp Glu Glu Asp Gly 990 995 CTACCGGCTC CGAGCCTGAG TTCTCTCTCCGGGGGCGGAG CCAACTCTCC CCCTTGTTGC 3091 TCTTACTACC ACCAATGAAA GCCATTAAAATGTCACTACC GAAAAAAAAA AAAAAA 3147 999 amino acids amino acid NotRelevant Not Relevant protein not relevant not relevant unknown 2 MetVal Gly Asp Ala Arg Pro Ser Gly Val Arg Ala Gly Gly Cys Arg 1 5 10 15Gly Ala Val Gly Ser Arg Thr Ser Ser Arg Ala Leu Arg Pro Pro Leu 20 25 30Pro Pro Leu Ser Ser Leu Phe Val Leu Phe Leu Ala Ala Pro Cys Ala 35 40 45Trp Ala Ala Gly Tyr Lys Thr Cys Pro Lys Val Lys Pro Asp Met Leu 50 55 60Asn Val His Leu Val Pro His Thr His Asp Asp Val Gly Trp Leu Lys 65 70 7580 Thr Val Asp Gln Tyr Phe Tyr Gly Ile Tyr Asn Asn Ile Gln Pro Ala 85 9095 Gly Val Gln Tyr Ile Leu Asp Ser Val Ile Ser Ser Leu Leu Ala Asn 100105 110 Pro Thr Arg Arg Phe Ile Tyr Val Glu Ile Ala Phe Phe Ser Arg Trp115 120 125 Trp Arg Gln Gln Thr Asn Ala Thr Gln Lys Ile Val Arg Glu LeuVal 130 135 140 Arg Gln Gly Arg Leu Glu Phe Ala Asn Gly Gly Trp Val MetAsn Asp 145 150 155 160 Glu Ala Thr Thr His Tyr Gly Ala Ile Ile Asp GlnMet Leu Arg Leu 165 170 175 Thr Arg Phe Leu Glu Glu Thr Phe Gly Ser AspGly Arg Pro Arg Val 180 185 190 Ala Trp His Ile Asp Pro Phe Gly His SerArg Glu Gln Ala Ser Leu 195 200 205 Phe Ala Gln Met Gly Phe Asp Gly PhePhe Phe Gly Arg Leu Asp Tyr 210 215 220 Gln Asp Lys Lys Val Arg Lys LysThr Leu Gln Met Glu Gln Val Trp 225 230 235 240 Arg Ala Ser Thr Ser LeuLys Pro Pro Thr Ala Asp Leu Phe Thr Ser 245 250 255 Val Leu Pro Asn MetTyr Asn Pro Pro Glu Gly Leu Cys Trp Asp Met 260 265 270 Leu Cys Ala AspLys Pro Val Val Glu Asp Thr Arg Ser Pro Glu Tyr 275 280 285 Asn Ala LysGlu Leu Val Arg Tyr Phe Leu Lys Leu Ala Thr Asp Gln 290 295 300 Gly LysLeu Tyr Arg Thr Lys His Thr Val Met Thr Met Gly Ser Asp 305 310 315 320Phe Gln Tyr Glu Asn Ala Asn Thr Trp Phe Lys Asn Leu Asp Lys Leu 325 330335 Ile Gln Leu Val Asn Ala Gln Gln Arg Ala Asn Gly Ile Arg Val Asn 340345 350 Val Leu Tyr Ser Thr Ser Ala Cys Tyr Leu Trp Glu Leu Asn Lys Ala355 360 365 Asn Leu Ser Trp Ser Val Lys Lys Asp Asp Phe Phe Pro Tyr AlaAsp 370 375 380 Gly Pro Tyr Met Phe Trp Thr Gly Tyr Phe Ser Ser Arg ProAla Leu 385 390 395 400 Lys Arg Tyr Glu Arg Leu Ser Tyr Asn Phe Leu GlnVal Cys Asn Gln 405 410 415 Leu Glu Ala Leu Ala Gly Pro Ala Ala Asn ValGly Pro Tyr Gly Ser 420 425 430 Gly Asp Ser Ala Pro Leu Asn Glu Ala MetAla Val Leu Gln His His 435 440 445 Asp Ala Val Ser Gly Thr Ser Arg GlnHis Val Ala Asn Asp Tyr Ala 450 455 460 Arg Gln Leu Ser Glu Gly Trp ArgPro Cys Glu Val Leu Met Ser Asn 465 470 475 480 Ala Leu Ala His Leu SerGly Leu Lys Glu Asp Phe Ala Phe Cys Arg 485 490 495 Lys Leu Asn Ile SerIle Cys Pro Leu Thr Gln Thr Ala Glu Arg Phe 500 505 510 Gln Val Ile ValTyr Asn Pro Leu Gly Arg Lys Val Asp Trp Met Val 515 520 525 Arg Leu ProVal Ser Lys His Val Tyr Leu Val Lys Asp Pro Gly Gly 530 535 540 Lys IleVal Pro Ser Asp Val Val Thr Ile Pro Ser Ser Asp Ser Gln 545 550 555 560Glu Leu Leu Phe Ser Ala Leu Val Pro Ala Val Gly Phe Ser Ile Tyr 565 570575 Ser Val Ser Gln Met Pro Asn Gln Arg Pro Gln Lys Ser Trp Ser Arg 580585 590 Asp Leu Val Ile Gln Asn Glu Tyr Leu Arg Ala Arg Phe Asp Pro Asn595 600 605 Thr Gly Leu Leu Met Glu Leu Glu Asn Leu Glu Gln Asn Leu LeuLeu 610 615 620 Pro Val Arg Gln Ala Phe Tyr Trp Tyr Asn Ala Ser Thr GlyAsn Asn 625 630 635 640 Leu Ser Ser Gln Ala Ser Gly Ala Tyr Ile Phe ArgPro Asn Gln Asn 645 650 655 Lys Pro Leu Phe Val Ser His Trp Ala Gln ThrHis Leu Val Lys Ala 660 665 670 Ser Leu Val Gln Glu Val His Gln Asn PheSer Ala Trp Cys Ser Gln 675 680 685 Val Val Arg Leu Tyr Pro Arg Gln ArgHis Leu Glu Leu Glu Trp Thr 690 695 700 Val Gly Pro Ile Pro Val Gly AspGly Trp Gly Lys Glu Val Ile Ser 705 710 715 720 Arg Phe Asp Thr Ala LeuAla Thr Arg Gly Leu Phe Tyr Thr Asp Ser 725 730 735 Asn Gly Arg Glu IleLeu Glu Arg Arg Arg Asn Tyr Arg Pro Thr Trp 740 745 750 Lys Leu Asn GlnThr Glu Pro Val Ala Gly Asn Tyr Tyr Pro Val Asn 755 760 765 Ser Arg IleTyr Ile Thr Asp Gly Asn Met Gln Leu Thr Val Leu Thr 770 775 780 Asp ArgSer Gln Gly Gly Ser Ser Leu Arg Asp Gly Ser Leu Glu Leu 785 790 795 800Met Val His Arg Arg Leu Leu Lys Asp Asp Ala Arg Gly Val Gly Glu 805 810815 Pro Leu Asn Lys Glu Gly Ser Gly Leu Trp Val Arg Gly Arg His Leu 820825 830 Val Leu Leu Asp Lys Lys Glu Thr Ala Ala Ala Arg His Arg Leu Gln835 840 845 Ala Glu Met Glu Val Leu Ala Pro Gln Val Val Leu Ala Gln GlyGly 850 855 860 Gly Ala Arg Tyr Arg Leu Glu Lys Ala Pro Arg Thr Gln PheSer Gly 865 870 875 880 Leu Arg Arg Glu Leu Pro Pro Ser Val Arg Leu LeuThr Leu Ala Arg 885 890 895 Trp Gly Pro Glu Thr Leu Leu Leu Arg Leu GluHis Gln Phe Ala Val 900 905 910 Gly Glu Asp Ser Gly Arg Asn Leu Ser SerPro Val Thr Leu Asp Leu 915 920 925 Thr Asn Leu Phe Ser Ala Phe Thr IleThr Asn Leu Arg Glu Thr Thr 930 935 940 Leu Ala Ala Asn Gln Leu Leu AlaTyr Ala Ser Arg Leu Gln Trp Thr 945 950 955 960 Thr Asp Thr Gly Pro ThrPro His Pro Ser Pro Ser Arg Pro Val Ser 965 970 975 Ala Thr Ile Thr LeuGln Pro Met Glu Ile Arg Thr Phe Leu Ala Ser 980 985 990 Val Gln Trp GluGlu Asp Gly 995 11703 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 3 GCGGCTGCAG AGCCATGGTT GGTGACGCGC GGCCTTCAGGGGTTCGCGCT GGCGGCTGCC 60 GGGGCGCGGT AGGATCCCGG ACGAGCTCCC GCGCGCTGCGGCCACCGCTC CCGCCTCTCT 120 CCTCCCTCTT CGTGTTGTTC CTAGCGGCGC CCTGCGCTTGGGCGGCGGGA TACAAGGTGA 180 GCGCGGCCCG CTAGCGGAAA TGTACAAGAG CCATAGTGAAGCCTCCAGTA GAGTCGGAGG 240 TGTGTGCGTG GGTCTGTTTT GTGGGTGCCC AGTGAATGGTTGCTAATATG ACAGTGTGAT 300 CTGGTTCATG CTTTGTGTTA CTGAGAAGAC TGGCTGTGTTAGTCTGAGAA TGGGGCTGTC 360 TGTGTCTGTC TCTTGCTTCT GTGGATTGGC TTACCTGGACTTGGCAAGCA TTTACACGAG 420 CGGGCTGTGT GGTGGGGACT GGTTGAGAGT TGGGAGTCAGCTGCCTGAAG TTTTAACCTG 480 ACTTCTCAAC TTGTGACCTT GGCCAAATCA CNTCACTTCTCTGAGCCTCT GTATTCTCAT 540 CTGAAAACTG GAGATAATGT TGCCCTCAGG TCCCAGGGTGCCTGCTTGGT ATTAACAAAT 600 GCTTAATAAA CATGAGCTAC TACTAGTGTT TTCCGAGGGCATGAACGAGA GGTGCTCTGA 660 GAAGTTCTGT CAGTTGGGGA GTACATCTAT GACACAACTATGTGTGTCCT TCTTAGGGGA 720 GCCGATCCCC ACCTGTCACT TGCTTCTAGA TCAAGACTTACCTTATATCC TCCCAACCCN 780 CCACTGCAGC CTGCCTCTTA ACCTTGGAGT TACTGACAGAGTGAGTGTGT GTTTGGGGTC 840 CCTGTGCAGA CATGCCCGAA GGTGAAGCCG GACATGCTGAATGTACACCT GGTGCCTCAC 900 ACACATGATG ATGTAGGCTG GCTCAAGACG GTGGACCAGTACTTCTATGG CAGTGAGTAG 960 AGGAGGGTGG GGAGTGACCC CTGGGACTCC CATGGTCCTGCGGAGCCCTT AAAATTCCTT 1020 TTCAGGCCTG GACAATCAGG GTGGGGGCAA CACCCAGCTTGGGCTCCTGT GTCTAAGAAT 1080 GTTTCCCTTG GCTTGCTGAT TTCTGATTGN CTGACCCCTGTGCCCACAGT CTACAATAAC 1140 ATCCAGCCGG CGGGTGTACA GTACATCCTA GACTCCGTCATCTCTTCCTT GCTGGCGAAT 1200 CCCACCCGCC GCTTCATCTA TGTGGAAATC GCCTTCTTCTCGCGTTGGTG GCGCCAGCAG 1260 ACAAATGCAA CACAGAAAAT CGTGAGGGAA CTGGTGCGCCAGGGTGAGCC TCCCTTCAGG 1320 AAGTGAAAAG AGGAAGCCAA GCCCAGCTTC TATCTCTAGCACCCTGGCTT CTGAGATTTT 1380 ATCACGCCAT TTGCAGCCTC TATGTGGCTG CCGTTGCTGCTTCTGCTAAG TCGCTTCAAT 1440 CGTGTCCGAC TCTGTGTGAC CCCATAGACA GCAGCCCACGAGGCTCCCCC ATCCCTGGGA 1500 TTCTCCAGGC AAGAACACTG GAGTGGATTG TGTTCCATAGCTCTCTTACA CTGGCCTGAG 1560 AGTGACCCCT GACCCTTCTC CTCTCAGGCC TGGTCGTTTAAGCAGGTCTT CTCGTCCCTG 1620 GCATCACCAA CCCTGGCGCC ACTCCTGGCC CTGACAACTGACTTGGACTT TGCCCCTCCC 1680 GGCACAGGAC GCCTAGAGTT CGCCAACGGT GGCTGGGTGATGAACGATGA GGCGACCACC 1740 CACTACGGAG CCATCATTGA CCAGATGACA CTCAGACTGCGCTTCCTGGA GGAGACGTTC 1800 GGCAGCGACG GGCGCCCCCG TGTGGCCTGG CACATCGACCCATTCGGCCA CTCTCGGGAG 1860 CAAGCTTCAC TGTTCGCGCA GTGGCTGGGT GATGAACGATGAGGCGACCA CCCACTACGG 1920 AGCCATCATC GACCAGATGA CACTCAGACT GCGCTTCCTGGAGGAGACGT TCGGCAGCGA 1980 CGGGCGCCCC CGTGTGGCCT GGCACATCGA CCCATTCGGCCACTCTCGGG AGCAAGCTTC 2040 ACTGTTCGCG CAGGTTTTCA GATCTCTTGG GCCCGCCCCTTCATTCCTTC TGACTCCTCC 2100 TCTGTCATCC AAGCCCCGCC CTTTTCTGNA AGTTCACCCGAACCCGAACC AGGCCCTACC 2160 CCTGGNCCTC TCGCCACTTA AGACCCTGCC TCTTGGGTGACCTGTGAATC CCATTCTTTT 2220 NGGTCTGGCC TTGGTTCTGC TCTGTCCTAG NCTAGGTTGACCTCATCAAC TATTCCCATA 2280 CAACCCGGNC TCCCTTGTCA GGTGAGTNTC CCCCTCCCTGATCCANCCAG TTGGTCTGAT 2340 CTGGTNTTGG CAAGTGGTGG TTGTAGGGCT GGGTTTCANCAGTTCGTACT GTGCATACAC 2400 CCTCCTGTAG TNGGANGGAG CNCTGATGGA GNGGTGTGGGTGGTGTCCCG GTTCNAGGTN 2460 TACTCCAAAC ANCTTTCNTG NCTGCCTCCT TCCAACCGGGTNACCTAAAC AATCCAAAAC 2520 CCGGCNCCTT GCAATNATCT CCCCTCCCTG ATCAACAAGTTNTCTGACCT GCTCTTGCCC 2580 AACCTGGTGG CTGGTTAGGN CCTCGGTTTT TCAACCAACCTCGTTACCTG TNCCATGACC 2640 ATCCCCCTTC CCTTGCTAGG CTGCGGAAAG GGGAAGGCCTCCTAGANCTG GGGAAGGTGG 2700 AGGTGGGTTG TAGCGTTGGA CTTGCTCCCT CCCGGCGCTTCCGCAGGGCT TCTGACCTTC 2760 CTCAGCCTTT GAAATGAACT GGAGGGCCTC GCTGGTCTTTGACNTGGTTT TTCTCCCTGT 2820 GCGTGAGAGG TTGGTGGTTG GTGATGAGAG GACCGGTCCCTTATGCATCC TGCCCTCTCN 2880 TGNTCTCCCA NCCCACTCGT CATCCCTCCC CANCTCCAGATGGGTTTTGA CGGCTTCTTC 2940 TTTGGACGCC TGGATTATCA AGACAAGAAG GTGCGGAAAAAGACGCTGCA GATGGAGCAG 3000 GTGTGGCGGG CCAGCACCAG CCTGAAACCT CCCACTGCCGACCTCTTCAC CAGTAAGGTG 3060 GTAGAGTGGA AAGAGGSCCG CCCCCGTGCT CAGAAGGGCCCTGGGCTTGG TTTATGGTCT 3120 GCTATCATTG TCTTGAAATT CCTAGTAGTT TATGAACAGGGGCCCACCAT TTGSAGTTTG 3180 CACTGGGCCT GGCAAATTCT GAAATCCATC CCCGGTGAGGCCTGGYAGGT CTAGGGGCCA 3240 TGACCACCCC CTGAACCTAA TGTGGTCCGC AGGTGTGCTCCCCAACATGT ACAACCCGCC 3300 GGAAGGTCTG TGCTGGGACA TGCTGTGTGC CGACAAGCCGGTTGTGGAGG ACACGCGTAG 3360 CCCAGAGTAC AACGCAAAAG AGCTGGTCCG TTACTTCCTGAAGTTGGCCA CTGACCAGGT 3420 AACCGGGTGT CCAGAACCTA TGCCTCCAGT GTACACGCACTGGGCCCTTC CATCGGCCCA 3480 GACAATCCCT AGCACTTCCT CACCTTCACT GGGGGAAGGTAAAATTCCAT TCACCATCAC 3540 CATACCCTGC TCCTGGATTT GTGTGCATTT CTGATTAGAAAGGTGGAGCC CTTCGCCAGA 3600 GCACATCCCC ACCATGTTTG NTAGACAGCA TGGNACAGGACCTCAGTACC CATGTCTGGG 3660 TGGTAGGCCC AAGAGAATTC CTCAACCGCT TGTGGCTCTTTTTCTGTGTG TCCCTCTGCT 3720 CCCATGTGAC ACACTTCCAC CCCTACCCCC CATGGCTCTGTGCACTCACA TTCTTTTTAT 3780 TTAAAAAAAT AATTTTTGTT TTTGCCTGTA CTGGGTCCTTTGTTNCNAAT CCGNACAATT 3840 TTTCTAGTTT GGCCAAGTGG GGGCTACTCT CTAGTTGTGATACTTGGCCT TCTCATTACG 3900 GTGNCTTCTT TTTGTTGTGG AGCATGGNCT CTAAGGCTCTTGGGCTTCAG TAGTTGCACC 3960 TCCCAGCCTG TAGAGCACAG GCTTGAGAGC GTGGTGCAGGGCTTAGTTGC TCTGAGGCAT 4020 GTGGGATCTT CCCAGATCAG GGATCGAACC CATGTCTCCTGCATTGGCAG ACAGAGTCTT 4080 TACCACTGAG CCACCAGGGA AGCCCTGTGC ACTCACATTCTTGACCACAT ATATACCAAG 4140 ACACAGCTGT CCACAGGGGT GGCGCAGGAC ACCCTAGCCTTAGGATACCC CCATCTTGCC 4200 TGCAGGGTAA GCTCTACCGC ACCAAACACA CTGTGATGACCATGGGCTCA GACTTCCAGT 4260 ACGAGAATGC CAACACGTGG TTCAAAAATC TTGACAAGCTCATCCAGTTG GTCAATGCCC 4320 AGGTGAGTGT GCCTSCCCGT GGGMACTKGT WTTKGTWTCCCAGGGYTTKG GGTCACATAC 4380 ATTATCTATA GGTGCTRCCT TAGTTTTCTA TACTTAATAAGCTACCACAA ACTTAGTGGC 4440 TTAAAACAAT AGCAAGGATA TTACCTTACA GTTCTGTAGGTCACAAGTCT GACATGGGTC 4500 TCACTGGGCT AAAATCAAGA TGTTAGCAGG GCTGTGTTCTNCTGGAAGCT CTAGGGGAAA 4560 GTCTCCTTGG CTCATCCAGC TTCATTANTA NTCCCTNCCCNAGAATGTCA TATTTCAATT 4620 CTCCATNCAA GTTTTAAGTA ATAAAATTGG AATATTGAAAGTTTAGTAAA ATCTCAGGTT 4680 TATTCTTGCA TCCCTCAATT TCTCTCCAGG CCAGGCTGGTAATTAGCTTG GNCCAATGTT 4740 CATTTTCACA CTTAGCCGTT GGTTTGTACT TAAACTGTGTATTTAAAAAA AGAGAGAAAC 4800 TTTGNACCAA CCGGGTGCAT GAATGTGTGT GCCTGTGTTTGTGTGCATGT GCACACCTGT 4860 GGGCCCGCCC GGGAAGGGCT CCCGAGGGCT CACATAGGCACACCTCCCCT CAGCAACGGG 4920 CCAACGGGAT CCGCGTCAAT GTTCTCTACT CTACCTCGGCCTGTTACCTC TGGGAGCTGA 4980 ACAAGGCCAA CCTCAGCTGG TATTTGGGGG GACTGGGGAGCCTCGGGGGG TTGGCATGCC 5040 CTGTGGGTCG TGGCCCTGCC CCCAATGTCT CTGCTGCTGCAGGTCAGTGA AAAAGGATGA 5100 CTTCTTCCCC TATGCTGATG GCCCCTACAT GTTCTGGACCGGTTACTTTT CCAGCCGGCC 5160 TGCCCTCAAA CGCTACGAGC GTCTCAGCTA CAATTTCCTGCAGGTAGGTG GACGCCAGGC 5220 TCCAGGGGCT GGCCCAGGGG TCCTGACAGG ACTGGTGCCCCAACATACCA CCTGCTCCAC 5280 AGGTGTGCAA CCAGCTGGAG GCGCTGGCGG GTCCGGCAGCCAACGTGGGA CCCTATGGCT 5340 CCGGGGACAG TGCACCCCTC AGTAGGTGTC GGCGGGCGAGGGGACAGCGG GGTGGGACTG 5400 AAGCTGGACT CCAGACTTCT ACTGTCCCTT TCTTAAAGCCTTTAAGAACC CAGCCTGCCA 5460 GACTTTTCGC ATGTCCTTGG GGTCTGGGCC GAGAGTCCTGCGGAGACCTC ACTTAGGCTA 5520 CACCGTCTGG CTACAGATGA GGCGATGGCC GTGCTCCAGCACCATGATGC AGTCAGTGGT 5580 ACCTCCCGGC AGCACGTGGC TAACGACTAT GCCCGCCAACTTTCAGAAGG CTGGAGGCCT 5640 TGCGAGGTGT GAGGGTGGGG CCTGGGGGAG GCGGAGACAGGAAGGGACTG GACCTGGACA 5700 CGGGGGCCGG AAGGTGGTGG GGGGCGGTGG GTGGTGGGGGCGGGGGTGGG GTCGGACTTA 5760 GGAAGGGGCG TGGTCGAAAA ACAGCCCGCC AGAAGGGCTCGTGGGGCGGG GCTTGTGAAA 5820 GGAGGGACGG AGAGAGGCAG GGGCGGGGCT GAGAGCGGGATCGCGAGGAG ACCGGCAGGG 5880 GCGTCCAGAG TGGAACTTTT GCTTTACACG CCTCCCCAGGTTCTCATGAG CAATGCGCTG 5940 GCGCATCTCA GCGGCTTAAA GGAGGACTTC GCCTTTTGTCGCAAGCTCAA CATCAGCATT 6000 TGTCCACTCA CGCAGACAGC AGAGAGAGTG AGCCGGCCTGGAGCGGGAGA GGCGGGGCTG 6060 GGGGCAGGTC CCGTGGGTGG TGGGTGGAAC GAGAACCAGAGAAACCTCTG GGCCCAGTGA 6120 AAAGGAGGAG GGGCTGATGG GCTCAGCTGG TTACTGAGCACTCTGGGGCA AGATATTTGA 6180 GGGCTCAGTG AAGCTGGGGC AGGACGGGGA CCAATGCACCACTTGGAGGC TTGACTCACC 6240 CAGGGACGTT GCTGCATACG TGGGTGGGTT TGCAGAAGGCCTGTTGTGAC CTGTGTCGGC 6300 TCTGACCATC CCACCCCAGT TCCAGGTGAT CGTTTATAACCCCCTGGGGC GGAAAGTGGA 6360 CTGGATGGTG CGGCTGCCTG TCAGCAAACA CGTTTACCTCGTGAAGGACC CCGGTGGCAA 6420 AATTGTGCCC AGCGATGTTA ACCCATTTCC ACAAAATATCCCNCCCTGTG TGCCCACTTT 6480 AATAATACCA CCCCCTTGAA ACCCCCCTCA TGGCATTCTCCTTACCCATG ANATTCTAAA 6540 TAATTTTCCC TTCTGCTCCT TTAAATCATA ACCCCCTTGGGAACATTCCT GCCCCTCGCC 6600 TATGAGTATC TCCCCCTTGT TAATATCTCC CTGTGAAAAAATTTCCTGTT TATGAATATT 6660 CTCCTCATCC TGAGGACCTC TCCCTCCTAA CCACTTCCTCCCCCCAGAAT CTGTTTTTCC 6720 CTCCTTCCCC TGTCTCAAGA GTCTCTTCCC ACGCTTAACTCCCTCACTTC TCCCACTTCC 6780 TACGGTACGT CCACCCTTCT GGTAGATTTC CCACCTTCTGAATCTTGGAA TCTGTTTCTC 6840 TTTTTTTGGC CATGCAGCTG GCAAGATCTT AGTTCCCCCACCAGGGGTCA AACTNGTGTC 6900 CCCTGCAGTG GATGTGGAGT CTAACCACTG GACTGCCAGGGAATTCCCTC TTTGCCATCT 6960 TTAACTCAGC ATTTCTGACA CTCCCACCCT CTCTGCCCAACACCTAAATT TCTTTTCCCC 7020 ATCTGGCAGG TGTGACTCCT TGCCCTCCTC AAGTTTGACCCCTTCCTGTT TGTGTGGTGT 7080 GGCTTCATGC ATGTCCTCCC AGCTTCTTGA CTTGGTTTCTTCCTCCCTTA CCTGAGGTGG 7140 TGACCATTCC CAGTTCAGAC AGTCAGGAGC TGCTTTTCTCAGCCTTAGTG CCTGCCGTGG 7200 GCTTCAGCAT CTACTCAGTC TCCCAGATGC CTAACCAAAGACCCCAGAAG TCCTGGTCCC 7260 GTGACTTGGT CATCCAGAAT GAGGTGAGAC CCTACTCAGACCCCCTTCCA TTTCTGGGTG 7320 ATAGTTTTGA GATGTGGCAG TAAGCCACAT GGACTGTGGGTGAGTGGGCG TGAAGTTTAT 7380 GGTCTTGTGT CATCAGTCCT CCACTGTATG TTCTCAGTGTCCTCTCTTGG GGCTCTTATG 7440 TCACCCTTGG GTGACACTTG ATAGAAATGT CAGAGCTGATGGAGGTATGG GTTTGNAAAT 7500 TCAGTGAGGT GTGTCAGAGA CGTGGAGGAG GTAGCTGTGTTGGTCATTTG GGGGTAAGAG 7560 AGATCCAGTC AGGCAGGGAG CACCCTCAAG TTGGNAGGGTGTTGGGTTGT TCAAAAGACA 7620 NTCAATTGTG TTCTGTGGGT CCCTCNTTCA ATTTCACCAAANAANCCTGG GTCCCCAANA 7680 AGATGGAGAA GGNAAGGCCA TGGGAAGTGG GGAAGAAGTGGTCAAGATTG AGGATTAGGG 7740 AAGAAAATGG ACCTGTAAGA TTTCCTNCCA GTGATGCACAATGAGAGAGA GAGAGGAGGG 7800 AAGGGTGGGT GGACCTTATT TTCCCCACCA GGGATTGAACACTCTCCTTC TACCATGCAA 7860 GCTGTGAGTC TAAATCACCC GTCTGCCAGG GAATTCCCACTTTGCAGTCT TTAATTCAGC 7920 ATTTCAGACA TGCGATCCTT CCTGCCCAAC ACCAGTGTTTGCTCCTGATC CCTGGGAAGA 7980 ACCATTGGTT GAAGTGATGG TCATTAATGT CTGTCCACCCTTTTACTCCC CAGTACCTCC 8040 GGGCTAGGTT TGACCCTAAC ACAGGGCTCT TGATGGAGTTGGAGAACCTG GAGCAGAATC 8100 TCTTGCTGCC TGTTCGCCAA GCCTTCTACT GGTGAGAGAGGACCACCAGG TCAGGGGGTG 8160 GGGTGTGTGA ACAGAGCTGA GGTCCCTTGT CTGACTCTCACCTGCCCTGG CCCTAGGTAC 8220 AACGCCAGTA CAGGTAACAA CCTAAGCTCC CAGGCCTCCGGTGCCTACAT CTTCAGACCC 8280 AACCAGAACA AACCACTGTT CGTGAGCCAC TGGGCTCAGACCCACCTTGT GAAGGTCAGG 8340 GGGCTGAGAG TGGNNACTTG GGGAANGGGN GTNNAGGNTGGGGTGNATGT GGNGCGTGAT 8400 GTGCCTTGTG AGGGGTGGTG GGGAGAATTT ACATCTCCAATAGATAAGAA GGCTAAGACC 8460 AGAGGGGAAT ACTTGAGGAT TTTCACACAG GGTTGATAATCACAGCTGGT TGTATGACAG 8520 TTACACATGG AGGCCCATAG AAAGGCATTT CACATCTGTCGTGGGGATCA GGAGTGTACT 8580 TAGCTTTGGA AATTGGTGGG TAGGCTTGTT CCCATTTGGNCCCTGAATGC ACAGAGTCAA 8640 GTGTAACTTG CCTGAAAATT TCAATAGGGA TCTTCGATAGGAAGCCCCAN AATTTCCAAA 8700 AACCCTGGAA AGGACCATGG CAAANGCANN GGTTNAAANATAAAANCNCT TGACCACTAA 8760 CACAAATCTG GCACCAAGAT TNATCCACAC CCCAAAAACTTANTTGCCAT TGCTTGAGGA 8820 AAAAATCANT TNATGTTTTG TTGCCAAGCA CCNAACCTAGCAAGATAGGG GAGGTGGGCT 8880 GGCCCCCGAA CCTATTAAGT GGGGTTGACC CTGACGTAGGCCTTTGTGAT CTTCACANCT 8940 GGGGTGGACA TTTGCAGGGC TCACCTTTGC TCAGGTGCACGCTTACACCT GCCCCCACTC 9000 CCGTGTGCCC ACAGGCGTCC TTGGTGCAGG AAGTACACCAGAACTTCTCA GCCTGGTGTT 9060 CCCAGGTGGT TCGCCTGTAT CCCAGACAAC GGCACCTGGAGCTAGAGTGG ACAGTGGGGC 9120 CAATACCTGT GGGGTGAGCG GGGCTGGGGG CTGGGGGAAGGNCAAAGTGA GGTTAAAGTG 9180 AAGCTCACCA CCTTGCCATC CCATTGGGTA TAGAGACGGCTGGGGGAAGG AGGTCATCAG 9240 TCGCTTTGAC ACTGCATTGG CGACACGCGG ACTCTTCTACACTGACAGCA ATGGCCGGGA 9300 GATCCTGGAG AGGAGGTGGA GAGACTGGGG GCACCGAGGGGTGGTCTGTG GTGTGCTGGG 9360 GCCCAGGGCA GTGAGGGGGC ATCTGCTGAT CCTAATGACTGTGGGAGGGA GAGGATGAAG 9420 GAGGAGTGGG TGAGTGGGGG GAAGGGAACC AGACTCCAAGCCTGATCNAA TCCTAACCCC 9480 ACCCCAGGCG GAATTATAGA CCTACCTGGA AGCTGAACCAGACTGAACCC GTGGCTGGAA 9540 ATTACTATCC AGTCAACAGC CGCATTTACA TCACGGTACCCATCCCCCAC CCTGCTCCCC 9600 ACCTTTTCCT GACACCCCTT TACAGAGTGG ACTTCACCTGTTCTTGACAT CTCCCAACTG 9660 TCCTCAGCAG TCTCCACCAT CCCTGTGGGG CCTGCCTGGGAGCCGGGGCT GGCCAGCAGT 9720 GCAGCCCCTC ACTCACCCTT TACCCCTCAG GATGGGAACATGCAGCTGAC TGTGCTCACT 9780 GACCGGTCCC AGGGGGGCAG TAGCCTGAGA GATGGCTCCTTGGAACTCAT GGTTAGTGGT 9840 CTGAGCCCCC ATCTAAGTCA GGGTCCTTCC ACCAGTTCCCTTCCTGGCCT CTACAGGACT 9900 TGAGGCAGTT TCTTTTGGTA GGTGCACCCT TGGTTGNGGTCCCGCTAAGC TGAACCTCCA 9960 TCCTCTTGTG AGAAATCANT CCGGGTTTTT TCAATCCCTACCAAATTCCG TTCAGGCCTG 10020 ATTACCTATG ACCCTACCCA AACTTCCGCT CCAGGCCCTGAATTACCCTA TGCACGCCCG 10080 CAATAGAACC TTACCGCCGT CTTCCCTAAN CCGTTNTTAGGNCGGAACCC CACATTTACT 10140 GGGGAACCCT TACGTTCCCT TCGTCGTCAA CCGTTTCCCCCAANAATTTT TTTTTCCCTT 10200 GAAATCCCCA CGTACTTTCA CCCAGTTTCC GGCCCAGAAATTGGCTACAG GAACCCTCAC 10260 TCTTGGCCAC TCTCCCCGCA GGTCCACCGA AGGCTGCTGAAGGACGATGC ACGCGGAGTT 10320 GGGGAGCCGC TGAACAAGGA GGGGTCGGGG CTTTGGGTGCGAGGACGTCA CCTCGTGCTG 10380 TTGGATAAGA AGGAGACTGC GGCCGCCAGG CACCGGTTACAGGCGGAGAT GGAGGTCCTG 10440 GCCCCGCAGG TGGTGCTGGC TCAAGGTGGC GGCGCGCGGTATCGCCTCGA GAAAGCCCCA 10500 CGCACGCAGG TGANGAGGCN CGGCAGAAAG AGGACCCAANGAACGCCTGC NGAGNGAGGG 10560 GCGGAGTTGA GGGCNGGTTN TCCTAGGTAN AACTGAGNGCCACCTCGGCT TGNGGTTGAC 10620 CAGGCCTCCT GATCNGGGTT GAGCNCNCCC TTCTGAATTGTATCACCCCC TCCTGTANCG 10680 TCTCANCCCC GCCCCTCCCG GCTCAGCCCC GCCCCGCTCTTTCCCTCAGT TCTCTGGGCT 10740 CCGCCGCGAG CTGCCACCCT CGGTACGTCT GCTCACATTGGCCCGCTGGG GCCCGGAGAC 10800 ACTGCTGCTG CGCTTAGAGC ACCAGTTCGC CGTAGGGGAGGACTCGGGCC GGAACTTGAG 10860 CTCCCCGGTG ACCCTGGACT TGACGGTGAG GATAGAGATGGAAAGGAGAC TGGGGAGAGG 10920 AGGGAGGGGA AACCCCGCTT NGTNCCAACG CATCCGGGCCCCTTCACTGC CCGCAGAACT 10980 TGTTTTCCGC CTTCACCATC ACCAACCTGC GGGAGACCACGCTGGCGGCC AACCAGCTCC 11040 TGGCCTACGC CTCCAGGCTC CAGTGGACGA CGGACACGGGTAGGAGCCTG CCCGGAGCGG 11100 GGTGGCGGCC GGGGGTCCGG NGAGGGGGGC GGCGGNTCAGTGTGGGAGGT GCGGGAATGT 11160 TGACCCGGTC CAGGTATAGA GCTTGGAGGG TCGGAGTAAGTCGGTGTTCA GATTTAGGTT 11220 AGGGGTTTAA GGGAGTGATG TGGCTTGACA GTCCTTGAGGGTGGGACTTG TGGGTGTGAG 11280 GACAGTGCCC AGACACTGGG GAGAGATTTG AGCATCTGGGGGTGGTATCT AGGCTCTGGC 11340 AAACAGTTGA AGGGTCTGGG AGTGAGGNCC TGGGGAAAGATTTAAANCGG TATGTCTGTG 11400 TTCAGGGGAG GNCGCACAGA GGATGTTAAG NCGGAGGAAGTCTGCATCCN TCACTTCTCC 11460 CCTCCACCTC NCCAGGCCCC ACACCCCATC CTTCTCCTTCCCGTCCGGTG TCCGCCACCA 11520 TCACGCTGCA GCCCATGGAA ATCCGTACCT TCTTGGCTTCGGTCCAATGG GAAGAGGACG 11580 GCTAGACCCA CTGGATACAA GACTACCGGC TCCGAGCCTGAGTTCTCTCT CCGGGGGCGG 11640 AGCCAACTCT CCCCCTTGTT GCTCTTACTA CCACCAATGAAAGCCATTAA AATGTCACTA 11700 CCG 11703 296 base pairs nucleic acid singlelinear DNA (genomic) NO NO unknown 4 CGCTGGACAC CCTAGCCTTA GGATACCCCCGTCTTGCCTG CAGGGTAAGC TCTACCGCAC 60 CAAACACACT GTGATGACCA TGGGCTCAGACTTCCAGTAC GAGAATGCCA ACACGTGGTT 120 CAAAAATCTT GACAAGCTCA TCCAGTTGGTCAATGCCCAG GTGAGTGTGC CTCCCCGTGG 180 GCACTTGTAT TTGTATCCCA GGGCTTTGGGTCACATACAT TATCTATAGG TGCTGCCTTA 240 GTTTTCTATA CCTTAATAAG CTACCACAAACTTAGTGGCT TAAAACAATA GCAAGG 296 23 base pairs nucleic acid singlelinear other nucleic acid /desc = “oligonucleotide” NO NO unknown 5CGCTGGACAC CCTAGCCTTA GGA 23 34 base pairs nucleic acid single linearother nucleic acid /desc = “oligonucleotide” NO YES unknown 6 CCTTGCTATTGTTTTAAGCC TCTAAGTTTG TGGT 34 21 base pairs nucleic acid single linearother nucleic acid /desc = “oligonucleotide” NO YES unknown 7 CGCAGGACACCCTAGCCTTA G 21 22 base pairs nucleic acid single linear other nucleicacid /desc = “oligonucleotide” NO YES unknown 8 GGGCTGCGCG TGTCCTCCAC AA22 24 base pairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 9 CAGAAAATCG TGAGGGAACT GGTG 24 23 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 10 CGCAGGACAC CCTAGCCTTA GGA 23 29 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown Base /note= “N = inosine” Base /note=“N = inosine” Base 12 /note= “N = inosine” Base 15 /note= “N = inosine”Base 18 /note= “N = inosine” Base 21 /note= “N = inosine” Base 24 /note=“N = inosine” Base 27 /note= “N = inosine” 11 ATNTACAANA CNGTNCCNAANGTNAANCC 29 26 base pairs nucleic acid single linear other nucleic acid/desc = “oligonucleotide” NO NO unknown Base /note= “N = inosine” Base/note= “N = inosine” Base 12 /note= “N = inosine” Base 18 /note= “N =inosine” Base 21 /note= “N = inosine” Base 24 /note= “N = inosine” 12ACNGCCATNG CNTCATTNAG NGGNGC 26 19 base pairs nucleic acid single linearother nucleic acid /desc = “oligonucleotide” NO NO unknown 13 AGAGAGGCGGGAGCGGTGG 19 24 base pairs nucleic acid single linear other nucleic acid/desc = “oligonucleotide” NO YES unknown 14 CAGAAAATCG TGAGGGAACT GGTG24 20 base pairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 15 GTGGCGGCGG CGGCTGCAGA 20 22 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 16 GGGCTACGCG TGTCCTCCAC AA 22 19 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 17 CCACCGCTCC CGCCTCTCT 19 20 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 18 ACTGACAGAG TGAGTGTGTG 20 19 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 19 TCATCATGTG TGTGAGGCA 19 22 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”NO YES unknown 20 TCTCTTCCTT GCTGGCGAAT CC 22 24 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligonucleotide” NO YESunknown 21 CAGAAAATCG TGAGGGAACT GGTG 24 19 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligonucleotide” NO YESunknown 22 GGAACAGGAC GCCTAGAGT 19 21 base pairs nucleic acid singlelinear other nucleic acid /desc = “oligonucleotide” NO NO unknown 23CGCAGTCTGA GTGTCATCTG G 21 22 base pairs nucleic acid single linearother nucleic acid /desc = “oligonucleotide” NO YES unknown 24AAGACGCTGC AGATGGAGCA GG 22 20 base pairs nucleic acid single linearother nucleic acid /desc = “oligonucleotide” NO NO unknown 25 TCCACTCTACCACCTTACTG 20 22 base pairs nucleic acid single linear other nucleicacid /desc = “oligonucleotide” NO NO unknown 26 GGGCTACGCG TGTCCTCCAC AA22 22 base pairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 27 TTGTGGAGGA CACGCGTAGC CC 22 20 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 28 GAACCACGTG TTGGCATTCT 20 30 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 29 CCTTGCTATT GTTTTAAGCC TCTAAGTTTG 3023 base pairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 30 CAAGCTCATC CAGTTGGTCA ATG 23 22 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 31 TCCGCGTCAA TGTTCTCTAC TC 22 23 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 32 TTCACTGACC AGCTGAGGTT GGC 23 21 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 33 GAGGTACCAC TGACTGCATC A 21 22 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 34 CATGATGCAG TCAGTGGTAC CT 22 21 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 35 TTGTCGCAAG CTCAACATCA G 21 21 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 36 TCTCTGCTGT CTGCGTGAGT G 21 23 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 37 TCAGGAGCTG CTTTTCTCAG CCT 23 20 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 38 GGAGACTGAG TAGATGCTGA 20 24 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 39 TAGAAGGCTT GGCGAACAGG CAGC 24 19 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 40 GGTGCCTACA TCTTCAGAC 19 19 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 41 CCTCACAAGG CACATCACG 19 19 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”NO YES unknown 42 GCTCAGGTGC ACGCTTACA 19 22 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligonucleotide” NO YESunknown 43 CGGCCATTGC TGTCAGTGAG AA 22 20 base pairs nucleic acid singlelinear other nucleic acid /desc = “oligonucleotide” NO YES unknown 44AAATTACTAT CCAGTCAACA 20 20 base pairs nucleic acid single linear othernucleic acid /desc = “oligonucleotide” NO NO unknown 45 GGAGATGTCAAGAACAGGTG 20 23 base pairs nucleic acid single linear other nucleicacid /desc = “oligonucleotide” NO YES unknown 46 CAGCACGAGG TGACGTCCTCGCA 23 21 base pairs nucleic acid single linear other nucleic acid /desc= “oligonucleotide” NO YES unknown 47 GGAGGTCCTG GCCCCGCAGG T 21 21 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 48 GACGGTGAGG ATAGAGATGG A 21 22 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 49 GGAGCTGGTT GGCCGCCAGC GT 22 21 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO YES unknown 50 CGCCTCCAGG CTCCAGTGGA C 21 24 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 51 TCTGAACACC GACTTACTCC GACC 24 18 basepairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” NO NO unknown 52 CCTGAACACA GACATACC 18 26 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”NO NO unknown 53 GGCTTTCATT GGTGGTAGTA AGAGCA 26 10 amino acids aminoacid Not Relevant Not Relevant peptide not relevant not relevantinternal unknown 54 Xaa Xaa Xaa Val Asn Xaa Xaa Tyr Ser Thr 1 5 10 24amino acids amino acid Not Relevant Not Relevant peptide not relevantnot relevant internal unknown 55 Xaa Ile Tyr Lys Ala Val Leu Tyr Ser ThrPro Ala Val Val Thr Pro 1 5 10 15 Pro Lys Val Lys Met Met Asn Ala 20 10amino acids amino acid Not Relevant Not Relevant peptide not relevantnot relevant internal unknown 56 Xaa Leu Leu Pro Val Val Gln Ala Phe Tyr1 5 10 11 amino acids amino acid Not Relevant Not Relevant peptide notrelevant not relevant internal unknown 57 Xaa Xaa Tyr Lys Thr Xaa ProLys Val Lys Pro 1 5 10 16 amino acids amino acid Not Relevant NotRelevant peptide not relevant not relevant internal unknown 58 Xaa GlyXaa Ser Ala Pro Leu Asn Glu Ala Met Ala Val Leu Gln Asp 1 5 10 15 20base pairs nucleic acid single linear DNA (genomic) NO NO unknown 59GGGATACAAG GTGAGCGCGG 20 20 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 60 CCCTGTGCAG ACATGCCCGA 20 20 base pairsnucleic acid single linear DNA (genomic) NO NO unknown 61 TTCTATGGCAGTGAGTAGAG 20 20 base pairs nucleic acid single linear DNA (genomic) NONO unknown 62 GTGCCCACAG TCTACAATAA 20 20 base pairs nucleic acid singlelinear DNA (genomic) NO NO unknown 63 GTGCGCCAGG GTGAGCCTCC 20 20 basepairs nucleic acid single linear DNA (genomic) NO NO unknown 64CCCGGCACAG GACGCCTAGA 20 20 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 65 GTTCGCGCAG GTTTTCAGAT 20 20 base pairsnucleic acid single linear DNA (genomic) NO NO unknown 66 CCANCTCCAGATGGGTTTTG 20 20 base pairs nucleic acid single linear DNA (genomic) NONO unknown 67 CTCTTCACCA GTAAGGTGGT 20 20 base pairs nucleic acid singlelinear DNA (genomic) NO NO unknown 68 TGGTCCGCAG GTGTGCTCCC 20 20 basepairs nucleic acid single linear DNA (genomic) NO NO unknown 69CACTGACCAG GTAACCGGGT 20 20 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 70 TTGCCTGCAG GGTAAGCTCT 20 20 base pairsnucleic acid single linear DNA (genomic) NO NO unknown 71 CAATGCCCAGGTGAGTGTGC 20 20 base pairs nucleic acid single linear DNA (genomic) NONO unknown 72 CTCCCCTCAG CAACGGGCCA 20 20 base pairs nucleic acid singlelinear DNA (genomic) NO NO unknown 73 ACCTCAGCTG GTATTTGGGG 20 20 basepairs nucleic acid single linear DNA (genomic) NO NO unknown 74GCTGCTGCAG GTCAGTGAAA 20 20 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 75 TTTCCTGCAG GTAGGTGGAC 20 20 base pairsnucleic acid single linear DNA (genomic) NO NO unknown 76 TGCTCCACAGGTGTGCAACC 20 20 base pairs nucleic acid single linear DNA (genomic) NONO unknown 77 GCACCCCTCA GTAGGTGTCG 20 20 base pairs nucleic acid singlelinear DNA (genomic) NO NO unknown 78 CTGGCTACAG ATGAGGCGAT 20 20 basepairs nucleic acid single linear DNA (genomic) NO NO unknown 79GCCTTGCGAG GTGTGAGGGT 20 20 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 80 GCCTCCCCAG GTTCTCATGA 20 20 base pairsnucleic acid single linear DNA (genomic) NO NO unknown 81 AGCAGAGAGAGTGAGCCGGC 20 20 base pairs nucleic acid single linear DNA (genomic) NONO unknown 82 CCCACCCCAG TTCCAGGTGA 20 20 base pairs nucleic acid singlelinear DNA (genomic) NO NO unknown 83 GCCCAGCGAT GTTAACCCAT 20 20 basepairs nucleic acid single linear DNA (genomic) NO NO unknown 84CTTACCTGAG GTGGTGACCA 20 20 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 85 CCAGAATGAG GTGAGACCCT 20 20 base pairsnucleic acid single linear DNA (genomic) NO NO unknown 86 TACTCCCCAGTACCTCCGGG 20 20 base pairs nucleic acid single linear DNA (genomic) NONO unknown 87 CCTTCTACTG GTGAGAGAGG 20 20 base pairs nucleic acid singlelinear DNA (genomic) NO NO unknown 88 CTGGCCCTAG GTACAACGCC 20 20 basepairs nucleic acid single linear DNA (genomic) NO NO unknown 89CCTTGTGAAG GTCAGGGGGC 20 20 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 90 GTGCCCACAG GCGTCCTTGG 20 20 base pairsnucleic acid single linear DNA (genomic) NO NO unknown 91 TACCTGTGGGGTGAGCGGGG 20 20 base pairs nucleic acid single linear DNA (genomic) NONO unknown 92 TTGGNTATAG AGACGGCTGG 20 20 base pairs nucleic acid singlelinear DNA (genomic) NO NO unknown 93 TGGAGAGGAG GTGGAGAGAC 20 20 basepairs nucleic acid single linear DNA (genomic) NO NO unknown 94CCCACCCCAG GCGGAATTAT 20 20 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 95 TTACATCACG GTACCCATCC 20 20 base pairsnucleic acid single linear DNA (genomic) NO NO unknown 96 TACCCCTCAGGATGGGAACA 20 20 base pairs nucleic acid single linear DNA (genomic) NONO unknown 97 GGAACTCATG GTTAGTGGTC 20 20 base pairs nucleic acid singlelinear DNA (genomic) NO NO unknown 98 CTCCCCGCAG GTGCACCGAA 20 20 basepairs nucleic acid single linear DNA (genomic) NO NO unknown 99ACGCACGCAG GTGANGAGGC 20 20 base pairs nucleic acid single linear DNA(genomic) NO NO unknown 100 TTTCCCTCAG TTCTCTGGGC 20 20 base pairsnucleic acid single linear DNA (genomic) NO NO unknown 101 GGACTTGACGGTGAGGATAG 20 20 base pairs nucleic acid single linear DNA (genomic) NONO unknown 102 CTGCCCGCAG AACTTGTTTT 20 20 base pairs nucleic acidsingle linear DNA (genomic) NO NO unknown 103 ACGGACACGG GTAGGAGCCT 2020 base pairs nucleic acid single linear DNA (genomic) NO NO unknown 104ACCTCNCCAG GCCCCACACC 20

What is claimed is:
 1. A method for diagnosing or screening for bovineα-mannosidosis, comprising detecting, in nucleic acid samples fromcattle, the presence or absence of an α-mannosidosis causing mutation ina nucleic acid encoding bovine lysosomal α-mannosidosis (LAMAN), whereinsaid mutation is a T to C transition at position 975 of SEQ ID NO: 1 oris a G to A transition at position 677 of SEQ ID NO: 1, and wherein thepresence of the mutation is indicative of a diseased or carrier animal,thereby diagnosing or screening for bovine α-mannosidosis.
 2. A methodof detecting α-mannosidosis-causing mutations in cattle, comprisingdetecting, in nucleic acid samples from cattle, the presence or absenceof a base transition in a nucleic acid encoding bovine LAMAN, whereinsaid transition is an α-mannosidosis-causing mutation, and wherein saidtransition is a T to C transition at position 975 of SEQ ID NO: 1 or isa G to A transition at position 677 of SEQ ID NO: 1, thereby detectingα-mannosidosis-causing mutations in cattle.
 3. A method as claimed inclaim 1 wherein the α-mannosidosis causing mutation is said T to Ctransition.
 4. A method as claimed in claim 1 wherein the α-mannosidosiscausing mutation is said G to A transition.
 5. A method as claimed inclaim 1, 2 or 3, wherein the cattle are of the Angus or Angus-derivedbreed.
 6. A method as claimed in claim 1, 2 or 4, wherein the cattle areof the Galloway breed.
 7. A method as claimed in claim 1, wherein thenucleic acid sample is amplified prior to detection of the mutation. 8.A method as claimed in claim 1, wherein the method of detectingcomprises a step of digesting the nucleic acid samples with arestriction enzyme.
 9. A method as claimed in claim 8, wherein themutation is detected by restriction fragment length polymorphism (RFLP)analysis.
 10. A method as claimed in claim 8 or 9, wherein therestriction enzyme is Mnl T.
 11. A method as claimed in claim 8 or 9,wherein the restriction enzyme is Bsa HI.
 12. A method as claimed inclaim 1, wherein the nucleic acid samples are DNA samples.
 13. A methodas claimed in claim 1, wherein the nucleic acid samples are from theroots of hair taken from the cattle.
 14. An oligonucleotide primerselected from the group consisting of SEQ ID NOS. 5 to 10 for use in thedetection of α-mannosidosis causing mutations.
 15. An oligonucleotideprimer selected from the group consisting of SEQ ID NOS. 5 to 10 for usein amplification of nucleic acid prior to detection of anα-mannosidosis-causing mutation.
 16. An isolated nucleic acid fragmentencoding a mutated bovine LAMAN protein, wherein said nucleic acidfragment comprises the bovine LAMAN open reading frame of nucleotides 15to 3085 of SEQ ID NO: 1, with the exception that the open reading framehas a T to C transition at position 975 of SEQ ID NO: 1 or a G to Atransition at position 677 of SEQ ID NO: 1.